Although protein kinase C, an enzyme dependent on calcium, phospholipid and diacylglycerol, has been found in high levels in ovarian tissues, its biologic function is yet unknown. In initial studies on the role of this enzyme in regulating ovarian functions, we compared protein kinase C activity in subcellular fractions of porcine corpora lutea and medium follicles. Highest protein kinase C-specific activities were found in the cytosol, followed by microsomes and mitochondria for both follicles and luteal tissues. Solubilization of all membrane-containing fractions by 0.2% Triton X-100 was required for full expression (a 4-fold average increase) of protein kinase activity. Extraction of membrane fractions with 0.5 M NaCl or sonication in a hypotonic medium revealed that 90% of the total mitochondrial protein kinase C activity and 50% of the microsomal activity was tightly membrane-bound. Characterization of both cytosolic and Triton X-100 extracted membrane preparations of luteal tissue by diethylaminoethyl (DEAE)-cellulose chromatography revealed a single peak of protein kinase C activity eluting at 80 mM NaCl. Cytosolic fractions of corpora lutea contained 3 times more protein kinase C-specific activity than did cytosolic fractions of follicles. In contrast, mitochondria from medium follicles contained 30% more specific protein kinase C activity than did luteal mitochondria. These higher cytosolic levels of protein kinase C-specific activity in corpora lutea suggest that the enzyme may play an important role in the process of luteinization or in the regulation of luteal function.