Platelet-rich thrombi are resistant to lysis by tissue-type plasminogen activator (t-PA). Although platelet alpha-granules contain plasminogen activator inhibitor-1 (PAI-1), a fast-acting inhibitor of t-PA, the contribution of PAI-1 to the antifibrinolytic effect of platelets has remained a subject of controversy. We recently reported a patient with a homozygous mutation within the PAI-1 gene that results in complete loss of PAI-1 expression. Platelets from this individual constitute a unique reagent with which to probe the role of platelet PAI-1 in the regulation of fibrinolysis. The effects of PAI-1-deficient platelets were compared with those of normal platelets in an in vitro clot lysis assay. Although the incorporation of PAI-1-deficient platelets into clots resulted in a moderate inhibition of t-PA-mediated fibrinolysis, normal platelets markedly inhibited clot lysis under the same conditions. However, no difference between PAI-1-deficient platelets and platelets with normal PAI-1 content was observed when streptokinase or a PAI-1-resistant t-PA mutant were used to initiate fibrinolysis. In addition, PAI-1-resistant t-PA was significantly more efficient in lysing clots containing normal platelets than wild-type t-PA. We conclude that platelets inhibit t-PA-mediated fibrinolysis by both PAI- 1-dependent and PAI-1-independent mechanisms. These results have important implications for the role of PAI-1 in the resistance of platelet-rich thrombi to lysis in vivo.