Plasmin caused a modest and gradual increase in platelet cytosolic Ca2+, mediated through both Ca2+ mobilization and external Ca2+ entry. This response was associated with accelerated Ca2+ extrusion and protein tyrosine phosphorylation. Plasmin-enhanced external Ca2+ entry and Ca2+ extrusion (but not Ca2+ mobilization) were attenuated by the tyrosine kinase inhibitor, genistein. Plasmin inhibited the thrombin-evoked increase in cytosolic Ca2+ and also inhibited the Ca2+ response to the tethered peptide TRAP-6 of the thrombin receptor. Furthermore, plasmin inhibited the binding of 125I-labeled alpha-thrombin to platelets. The inhibitory effect of plasmin on the thrombin response shared some characteristics with the effect of protein kinase C stimulators but was not reversed by protein kinase C inhibitors. Plasmin did not change platelet cyclic nucleotides. These results suggest a dual effect of plasmin. Plasmin produces a small rise in platelet cytosolic Ca2+ and a tyrosine kinase-dependent enhancement of Ca2+ turnover (external Ca2+ influx and Ca2+ efflux). However, it also attenuates the thrombin-evoked cytosolic Ca2+ response by blocking Ca2+ mobilization and slowing the rate of external Ca2+ influx. The latter feature would result in a plasmin-induced inhibition of thrombogenesis.