The hypothesis that protein kinase C may be an important regulator of ovarian theca-interstitial cell steroidogenesis was tested by using phorbol-12-myristate-13-acetate (PMA) and phorbol-12, 13-dibutyrate (PDB) to directly stimulate protein kinase C activity. Collagenase-dispersed cells (4 x 10(5) viable cells/dish) form ovaries of hypophysectomized immature rats were cultured in serum-free medium in the presence and absence of 0-100 ng/ml of luteinizing hormone (LH), PMA (0-100 nM), and/or PDB (0-100 nM). Treatment with 100 ng/ml LH stimulated androsterone production 100-fold at Day 4 of culture. The presence of 100 nM PMA or PDB had no effect on basal androsterone production; however, treatment with increasing concentrations of PMA or PDB (0-100 nM) caused a dose-related inhibition (maximum 70%) of LH-stimulated androsterone synthesis (ID50 = 1.8 nM and 2.4 nM, respectively). PMA and PDB did not significantly alter DNA, protein, or cell viability, indicating that their inhibitory effects were not due to changes in cell number or viability. Cells treated with LH and 100 nM 4 alpha-phorbol didecanoate (4 alpha-PDD; a phorbol ester that does not activate protein kinase C) failed to show significant decreases in androsterone production. Time-course studies revealed that when PMA treatment was delayed until Day 2 or 4 of culture, dramatic inhibitory effects on LH-stimulated androsterone production were still observed. These results suggest that the biological activity of protein kinase C is retained after the cells have expressed their differentiated state.(ABSTRACT TRUNCATED AT 250 WORDS)