The treatment of mouse eggs with phorbol esters and diacylglycerol inhibits sperm penetration and results in biochemical modification of the zona pellucida. In this report, we have demonstrated the presence of protein kinase C (PKC) activity in mouse eggs as determined by 12-O-tetradecanoyl phorbol-13-acetate (TPA) dependent in vivo and in vitro protein phosphorylation in mouse eggs. When mouse eggs were radiolabeled with [32P]phosphate and treated with TPA, two specific proteins, 70 and 20 kDa, were phosphorylated. The 70-kDa protein was also phosphorylated in vitro by endogenous PKC. In addition, we have shown that exogenous PKC induced the in vitro phosphorylation of 70-, 55-, and 20-kDa proteins in egg extract. The 70-kDa protein was also phosphorylated in vitro after treatment of the cytosol fraction of mouse eggs with TPA, suggesting that this protein might be a specific substrate for PKC and that it is located in the cytosol. These results demonstrate that mouse eggs contain PKC activity and suggest that PKC-catalyzed protein phosphorylation of specific proteins might be involved in the regulation of egg-induced modification of the zona pellucida.