Steroidogenic acute regulatory (StAR) protein plays a crucial role in the regulation of cholesterol transport from the outer mitochondrial membrane to the inner membrane, where P450scc participates in a rate-limiting step of adrenal steroidogenesis. We have already reported that both of cAMP- and protein kinase C-dependent processes may play a crucial role in the regulation of expression of StAR protein when bovine fasciculata cells are stimulated with ACTH. In the present study, ACTH increased cytosolic calcium movement and activated expression of StAR protein, resulting in enhancing cortisol production by bovine adrenal fasciculata cells. The role of the calcium/calmodulin-dependent protein kinase process in the regulation of expression of the StAR protein by ACTH was studied. The activating effects of ACTH on the StAR protein and cortisol production were inhibited by pretreatment with KN-93, a specific inhibitor of calcium/calmodulin-dependent protein kinase II. These findings suggest that ACTH can enhance expression of the StAR protein as well as cortisol synthesis in bovine adrenal fasciculata cells, in part via a calcium/calmodulin-dependent protein kinase process.