Staphylokinase is a highly fibrin-specific clot-dissolving agent that constitutes a promising drug for clinical development. It is of bacterial origin, and the majority of patients develop neutralizing Ab after its administration. Several antigenic regions, recognized by these Ab, have been identified, but the underlying immunogenic features of staphylokinase remain unknown. In this study, we show that staphylokinase is a T cell-dependent Ag, and that an immunological memory may be acquired, even without administration of staphylokinase. Thrombolysis with staphylokinase provokes the proliferation of staphylokinase-specific T lymphocytes, which remain elevated over 10 mo posttreatment. Interestingly, analysis of a large number of staphylokinase-specific T cell clones isolated from 10 unrelated donors revealed only six distinct immunogenic regions in the molecule. Moreover, five of the six regions are recognized by T lymphocytes from several individuals, indicating that these regions are not restricted to a single HLA-DR allele. Therefore, these new insights can guide the design of variants with a lower immunogenic profile in humans.