The effects of two protein kinase C (PKC) inhibitors, calphostin C and staurosporine, on the in vitro ovulation of goldfish (Carassius auratus) oocytes were investigated. Ovulation was stimulated by prostaglandin (PG) F2 alpha (PGF2 alpha, 2.0 micrograms/ml), by sodium orthovanadate (0.1 mM), by a combination of the phorbol ester phorbol 12-myristate-13-acetate (PMA, 0.1 micrograms/ml) and calcium ionophore A23187 (0.05 micrograms/ml), by thapsigargin (0.2 micrograms/ml), and by elevated pH (8.1). In addition, the effects of these inhibitors on the PKC activity of the goldfish follicle wall was determined by use of a specific peptide substrate phosphorylation assay. At 0.1 microM, staurosporine significantly blocked ovulation induced by all agents. However, at lower (0.01 microM) levels it blocked only PMA/A23187-induced ovulation. In contrast, calphostin significantly blocked only PMA/A23187-induced ovulation, although there was a decrease in pH-induced ovulation at lower calphostin concentrations. Both calphostin and staurosporine blocked follicular PKC activity at levels that were inhibitory to ovulation. In addition, staurosporine significantly blocked PKC activity at levels even lower than those needed to block ovulation. The combined results suggest that orthovanadate, PGF2 alpha, and thapsigargin do not require PKC activation for the induction of ovulation, whereas PMA/A23187 does.