We have studied the effect of ethoxzolamide , a specific carbonic anhydrase inhibitor, on the velocity of thrombin-stimulated platelet aggregation. After preincubation of platelet rich plasma with 10(-6) M ethoxzolamide the velocity of platelet aggregation was reduced by about 40%. Between 10(-11) M and 10(-10)M ethoxzolamide was necessary to achieve a half-maximal diminution of the aggregation velocity. An identical maximal reduction of the velocity of aggregation as with ethoxzolamide could be achieved by a nearly complete removal of CO2 from the platelet rich plasma. These results suggest that the intracellular CO2 hydration-dehydration reaction is involved in the activation of human platelets by thrombin. It is possible that the cytosolic carbonic anhydrase of platelets provides a rapid source of the protons that are transferred across the plasma membrane during the activation process.