Vascular endothelial and smooth muscle cells were shown to produce 2-AG on stimulation with either thrombin, the Ca2+ ionophore A23187  or carbachol . Apart from contributing to the formation of hypotensive 2-AG, blood macrophages (and platelets), if present near the vascular wall and capable of inactivating the monoglyceride, might also limit or terminate 2-AG action. For example, enzymatic hydrolysis is a limiting factor for the potency of the vasodilatory effect of anandamide in the mesenteric artery , and is likely to be important also for 2-AG. Therefore, we carried out a series of experiments aimed at assessing whether intact, living J774 macrophages can inactivate exogenous [3H]2-AG. We found that the amounts of [3H]2-AG incubated with cells decreased with an half-life varying between 19 and 28 min depending on the cell density and on the initial concentration of the compound (Fig. 3a and data not shown). In cell extracts, increasing amounts of radioactivity with increasing incubation times were found associated with AA and phospholipids in a temperature-sensitive manner. Unprocessed [3H]2-AG was also found whose levels were only slightly decreased at 0–4 °C. These findings are in agreement with analogous data reported previously for rat basophilic leukaemia RBL-2H3 cells ( and Di Marzo, unpublished data). In addition, we found that some radioactivity was also associated with diacylglycerol and triacylglycerols. It is possible that [3H]AA produced from [3H]2-AG hydrolysis is partly esterified into phosphoglycerides, as previously shown for anandamide in rat central neurons . However, when we tested several enzyme inhibitors, we observed that the incorporation of radioactivity into phospholipids and AA was inhibited to a different extent by some of these substances (Fig. 3b). In particular, [3H]AA, but not [3H]phospholipid, formation was inhibited by 100 µmp-hydroxy-mercuribenzoate (HO-BzHgOH). Conversely, 100 µm thimerosal significantly inhibited the formation of [3H]phospholipid (as well as [3H]diacylglycerols and [3H]triacylglycerols, data not shown) without increasing the amount of [3H]AA, and so did 100 µm PhCH2SO2F. 1-linoleoyl-glycerol was more potent as inhibitor of [3H]phospholipid than [3H]AA formation. The FAAH inhibitors CH2Δ4AchPOF and Δ4AchCoCH2F3 were, respectively, ineffective or weakly active against both processes. Interestingly, of the compounds tested, none significantly affected the levels of [3H]2-AG found in cell extracts (data not shown), including 1-linoleoyl-glycerol, which was previously found to inhibit 2-AG diffusion into RBL-2H3 and N18TG2 cells . On the basis of these findings, it is possible to hypothesize that 2-AG, once taken up by macrophages, is catabolized through several parallel enzymatic reactions, i.e. hydrolysis to AA and esterification into phospholipids, diacylglycerols and triacylglycerols. Clearly, our data do not allow to assess to what extent radiolabelled phosphoglycerides are produced from the direct esterification of [3H]2-AG or of [3H]AA produced from [3H]2-AG hydrolysis. As to whether the monoglyceride is taken up through passive or facilitated diffusion, we analysed the possibility that the same mechanism previously shown to facilitate anandamide transport inside RBL-2H3 and J774 cells [13,17] also recognizes 2-AG as substrate. This possibility was recently suggested by a study carried out in human astrocytoma cells . However, three sets of experimental data argue against 2-AG being a ligand for the anandamide ‘carrier’ in J774 macrophages. Firstly, the effect of decreasing the temperature of incubation to 4 °C on the amounts of [3H]2-AG found in J774 cell extracts was not comparable in its extent to that observed for [3H]anandamide uptake by the same or other cells [13,18,22,31]. In fact, the little effect observed (Fig. 3a) may have been due to the strong inhibition of [3H]2-AG hydrolysis or esterification to phospholipids which may result in augmented amounts of unprocessed [3H]2-AG, thus preventing the gradient-dependent diffusion of the monoglyceride into the cells. Secondly, 100 µm PhCH2SO2F, which inhibits anandamide re-uptake from RBL-2H3 and J774 cells  as well as U937 macrophages , did not affect the levels of [3H]2-AG found inside J774 cells (89.1 ± 13.5% of control). Finally, also 100 µm anandamide did not significantly reduce the levels of [3H]2-AG inside cells (88.0 ± 10% of control), nor did 50 µm 2-AG reduce the uptake of 3 nm[3H]anandamide from J774 cells (104.7 ± 2.2% of control, means ± SD, n = 3). In RBL-2H3 cells, where the facilitated transport mechanism for anandamide has been well characterized and is counteracted by several inhibitors [13,32], 50 µm 2-AG did not affect the uptake of 4 µm[14C]anandamide  but it did reduce the uptake of 3 nm[3H]anandamide, although only to a little extent (67.6 ± 5.5% of control, n = 3). These data suggest that, in J774 (and RBL-2H3) cells, 2-AG and anandamide are transported into cells through distinct mechanisms.