Endogenous 2-arachidonoylglycerol (2-AG) is antiinvasive in androgen-independent prostate carcinoma (PC-3) cells. Invasion of PC-3 cells is also inhibited by exogenously added noladin ether, a non-hydrolyzable analog of 2-AG. In contrast, exogenous 2-AG has the opposite effect. Cell invasion significantly increased with high concentrations of exogenous 2-AG. In PC-3 cells, arachidonic acid (AA) and 12-hydroxyeicosatetraenoic acid (12-HETE) concentrations increased along with exogenously added 2-AG, and 12-HETE concentrations increased with exogenously added AA. Invasion of PC-3 cells also increased with exogenously added AA and 12(S)-HETE but not 12(R)-HETE. The exogenous 2-AG-induced invasion of PC-3 cells was inhibited by 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP, an inhibitor of 2-AG hydrolysis) and baicalein (a 12-LO inhibitor). Western blot and RT-PCR analyses indicated expression of 12-HETE producing lipoxygenases (LOs), platelet-type 12-LO (P-12-LO) and leukocyte-type 12-LO (L-12-LO), in PC-3 cells. These results suggest that exogenous 2-AG induced, rather inhibited, cell invasion because of its rapid hydrolysis to free AA, and further metabolism by 12-LO of AA to 12(S)-HETE, a promoter of PC cell invasion. The results also suggest that PC-3 cells and human prostate stromal (WPMY-1) cells released free AA, 2-AG, and 12-HETE. In the microenvironment of the PC cells, this may contribute to the cell invasion. The 2-AG hydrolysis and concentration of 2-AG in microenvironment are critical for PC cell's fate. Therefore, inhibitors of 2-AG hydrolysis could potentially serve as therapeutic agents for the treatment of prostate cancer. © 2007 Wiley-Liss, Inc.