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Although the role of arachidonic acid (AA) in the regulation of steroidogenesis is well documented, the mechanism for AA release is not clear. Therefore, the aim of this study was to characterize the role of an acyl-CoA thioesterase (ARTISt) and an acyl-CoA synthetase as members of an alternative pathway in the regulation of the intracellular levels of AA in steroidogenesis. Purified recombinant ARTISt releases AA from arachidonoyl-CoA (AA-CoA) with a Km of 2 micro m. Antibodies raised against recombinant acyl-CoA thioesterase recognize the endogenous protein in both adrenal tissue and Y1 adrenal tumor cells by immunohistochemistry and immunocytochemistry and Western blot. Stimulation of Y1 cells with ACTH significantly stimulated endogenous mitochondrial thioesterases activity (1.8-fold). Nordihydroguaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochondrial acyl-CoA thioesterases. ACTH-stimulated steroid synthesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA-CoA synthetase. The apparent IC50 for NDGA was reduced from 50 micro m to 25, 7.5 and 4.5 micro m in the presence of 0.1, 0.5 and 2 micro m triacsin C, respectively. Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells.