The activity of protein kinase C (PKC) and the presence of endogenous PKC-modulatory factors in monkey CL have not been previously reported. The specific activity of Ca(2+)- and lipid-dependent PKC activity in cytosols prepared from CL obtained from rhesus monkeys at midluteal phase was 515.1 +/- 79 pmol/min x mg; Ca(2+)-independent PKC activity was 233.2 +/- 31 pmol/min x mg. No PKC-inhibitory activity was seen when luteal cytosol was mixed with a control preparation of PKC (control PKC 10.2 +/- 2.0 pmol/min; luteal cytosol plus control PKC 26.0 +/- 2.1 pmol/min). However, the monkey CL contained PKC-phosphatase activity (the endogenous inhibitor of PKC in the rat ovary is a protein phosphatase). Specific activity of luteal PKC phosphatase activity was 609.0 +/- 94 pmol/min x mg. Kinetic studies suggested that the monkey CL contained a competitive inhibitor of the phosphatase. Heat-treated cytosol mixed with control PKC (9.0 +/- 0.7 pmol/min) resulted in enzyme activity that was significantly greater than that of the control (13.7 +/- 1.2 pmol/min). Luteal cytosol that had been heat-treated and extracted with petroleum ether substituted for lipids in the activation of PKC. In summary, the activity of PKC in the monkey CL was readily measurable; it was not masked by an endogenous PKC-inhibitory factor. The monkey CL did contain a heat-stable PKC-stimulatory activity.