The effects of trypsin, acrosin and a recently described trypsin-like protease from bovine sperm were studied on adenylate cyclase activity in membranes of human platelets. These proteases caused an immediate decrease in adenylate cyclase activity, which was independent of the platelet membrane concentration used and which was constant for up to 20 min of incubation at 25 degrees C. When the incubation was prolonged, the proteases eliminated their own inhibitory action as well as that of the inhibitory hormone epinephrine. The adenylate cyclase inhibition caused by the proteases was strictly dependent on the presence of GTP (EC50 approximately 0.1 microM), whereas in the absence of GTP only minor changes in enzyme activity were observed at the conditions and protease concentrations used. Maximal inhibition caused by the proteases was between 40% and 60%. Half-maximal inhibition by the purified proteases trypsin and acrosin was observed at about 30 ng/ml and 2 micrograms/ml respectively. Inhibition of platelet adenylate cyclase by the proteases was partially additive with that caused by epinephrine, while with thrombin no additivity was observed. The serine protease inhibitor leupeptin blocked the actions of the proteases when added simultaneously with the enzymes, but was ineffective when added later on. Treatment of platelet membranes with the alkylating N-ethylmaleimide at low concentrations and Mn2+ ions (greater than or equal to 1 mM), both agents known to abolish inhibition of adenylate cyclase via the inhibitory guanine-nucleotide-binding protein Gi, eliminated the inhibitory action of the proteases. The data indicate that trypsin and trypsin-like proteases have two opposite effects on the platelet adenylate cyclase system, the well-documented elimination of Gi action and, as shown here, an immediate activation of Gi with subsequent adenylate cyclase inhibition. The data are consistent with the hypothesis that the activation of Gi caused by the proteases is due to an interaction of the proteases with specific cell-surface receptor sites in a manner similar to thrombin.