Purified rat Leydig cell cytosol was found to contain a protein kinase which was dependent on the presence of both calcium and phospholipids (phosphatidylserine and diolein), i.e. calcium/phospholipid-dependent protein kinase. The peak of Ca/phospholipid-dependent protein kinase was separated from type I and type II cAMP-dependent protein kinase by DE-52 chromatography. 4β-Phorbol-12-myristate-13-acetate (PMA), a tumor-promoting agent, could substitute for diolein in activation of Ca/phospholipid-dependent protein kinase. PMA caused dose-dependent increments of testosterone formation by Leydig cells, whereas inactive phorbol esters had no significant effects. PMA-induced testosterone formation was dependent on extracellular calcium and could be blocked by the addition of the calcium channel-blocking agent nifedipine. Since PMA can directly activate Ca/phospholipid-dependent protein kinase and increase testosterone formation, these results suggest that Ca/ phospholipid-dependent protein kinase may be involved in modulating Leydig cell steroidogenesis in addition to the classical cAMP-dependent protein kinase pathway. (Endocrinology117: 119–126, 1985)