Induction of an immune response is strongly dependent on the phylogenetic distance between antigen and recipient. In general, antibodies will not be raised against self-antigens nor against highly conserved domains. In the present study we describe the production and characterization of murine monoclonal "auto-antibodies" against murine tissue-type plasminogen activator (t-PA) raised in "knock-out" mice, homozygously deficient of the functional gene. 203 stable hybridomas were obtained producing murine monoclonal antibodies against murine t-PA. Analysis of the species reactivity revealed that 182 cross-reacted with one or more (t-)PAs originating from other species including rat t-PA, human t-PA, and vampire bat-PA. 121 reacted with epitopes conserved among murine, rat, and human t-PA. In addition, 31 of the monoclonal antibodies were directed against domains present in all four species. Epitope mapping indicated a high frequency of specificity toward diverse epitopes that are highly conserved across species. Comparative analysis of their influence on the enzymatic activity of t-PA and their species cross-reactivity clearly demonstrated that the domains required for the biological activity of plasminogen activators are more conserved (p < 0.02) than non-functional domains. The availability of such unique antibodies against a wide variety of conserved epitopes may facilitate studies on the structural homologies between (t-)PAs isolated from various species. The present approach should also apply to various other classes of proteins, allowing the generation of monoclonal antibodies, against conserved epitopes, which could not be raised in wild-type animals because of their "self-antigen" nature.