Various serine proteases (e.g., trypsin, alpha-chymotrypsin, Pronase, and subtilisin) stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in a membrane-enriched fraction of the rat ovary. Maximum stimulation is observed at protease concentrations ranging from 3 to 10 mug/ml. Higher protease concentrations inhibit ovarian adenylate cyclase in a dose-dependent manner. Protease stimulation causes a 6- to 8-fold increase in adenylate cyclase activity, which is comparable to the stimulation observed with human chorionic gonadotropin. Combinations of trypsin plus hormone or trypsin plus NaF stimulate ovarian adenylate cyclase activity to a greater extent than does any one of these alone. The mechanism of protease stimulation of adenylate cyclase involves limited proteolysis because zymogen precursors fail to activate the cyclase as does trypsin pretreated with trypsin inhibitors. Unlike cholera toxin, the serine protease stimulation is immediate (within the first 5 min) and requires no additional factors (e.g., NAD(+)). It is unlikely that protease stimulation of adenylate cyclase results from a proteolytic modification of the hormone receptor on the cell surface, because of the additive effects noted above and because protease stimulation is also observed in ovaries desensitized to hormone that lack this hormone receptor. Results with Lubrol-treated membranes also suggest that proteolytic enzymes do not directly activate the catalytic subunit of the cyclase or unmask new catalytic sites because the protease effect (like hormonal stimulation) is abolished by the detergent, whereas fluoride stimulation is enhanced. Other data suggest that serine protease and chorionic gonadotropin stimulation of adenylate cyclase result from activation of a membrane protease that then regulates adenylate cyclase in the ovary.