To examine the basis of enhanced thrombolytic effect of tissue-type plasminogen activator (t-PA) in the presence of lys- or glu-plasminogen, studies were performed with human platelet-rich plasma (PRP) and washed platelets (WP). t-PA inhibited platelet aggregation in PRP and this effect was potentiated by lys-plasminogen as well as glu-plasminogen. t-PA inhibited WP aggregation only in the presence of lys- or glu-plasminogen. The potentiation of the effects of t-PA was greater (P < 0.05) with lys-plasminogen than with glu-plasminogen. t-PA alone also decreased 14C-serotonin release from WP, and lys- as well as glu-plasminogen reversed this effect of low concentrations of t-PA in WP. Aggregation of WP was also inhibited by plasmin, a proteolytic product of plasminogen. Low, but not high concentrations, of plasmin increased the release of 14C-serotonin. Anti-aggregatory effects of plasmin and lys-plasminogen plus t-PA on platelets were attenuated by preincubation of PRP or WP suspension with aprotinin. These observations suggest enhanced inhibitory effect of t-PA on platelet function in the presence of lys-plasminogen as potential basis of salutary interaction in models of arterial thrombosis.