Abstract: Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 184.108.40.206) synthesis activity in the microvasculature of rat brain. Both ara-chidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 μM and 5.8 μM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol min−1 mg protein−1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 μM unlabelled arachidonic acid, with a Ki of 7.1 μM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 μM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 μM unlabelled palmitic acid, with a Ki of 5.0 μM and to a much lesser extent by arachidonic acid (Ki, 23 μM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40°C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis. These data together strongly suggest that rat brain microvessels have the capacity to catalyze specifically the formation of acyl-CoA derivatives from several polyunsaturated long-chain fatty acids, including arachidonic acid in the first place. Besides this particular arachidonoyl-CoA synthetase, palmitic acid could be activated with the aid of a second acyl-CoA synthetase.