We have isolated various phospholipids from adrenal mitochondria of adrenocorticotropic hormone (ACTH)-treated (stimulated) and cycloheximide/ACTH-treated (unstimulated) rats. When the effects of these phospholipids were examined on the formation of pregnenolone from endogenous cholesterol by adrenal mitochondria of unstimulated rats, phosphatidylethanolamine and phosphatidylserine from stimulated mitochondria were effective in enhancing the cleavage reaction in unstimulated mitochondria, whereas these phospholipids from unstimulated mitochondria were all ineffective. Cardiolipins from both stimulated and unstimulated mitochondria were effective. When the compositional changes in fatty acid moiety of phospholipids were examined, a significant increase in C22:4 (adrenic) acid was observed only for phosphatidylethanolamine under the influence of ACTH. A linear relationship between the contents of C22:4 acid in various phospholipids and respective steroidogenic activities was obtained (r = 0.880), suggesting an important role of this fatty acid moiety. The separation of active phosphatidylethanolamine by high performance liquid chromatography revealed that a fraction containing 25% C22:4 acid was most effective in the activation. Based on these results, it is most likely that 1-stearoyl-2-adrenoyl phosphatidylethanolamine is an active species. C22:4 acid was liberated together with C20:4 acid from adrenal triglycerides by the action of ACTH but the liberation was insensitive to cycloheximide inhibition. Finally, cardiolipin which enhances the transfer of cholesterol to cytochrome P-450scc may not be a physiological mediator of ACTH action.