Summary Antibodies were produced in goats by immunization with highly purified fibrinolysin(s) secreted by rat ovarian and breast carcinoma tumor cells in culture. The fibrinolysin had previously been shown to be different than a secreted plasminogen activator and biochemically distinct from plasmin in human, horse, and calf sera. After fractionation of the goat antiserum to eliminate activatable fibrinolytic factors and inhibitors, the antibodies in the isolated IgG fraction were characterized as 7S heat-stable (56°C, 30 min) non-precipitating types that neutralized fibrinolytic activity. Three assay methods were used to test the inhibitory effect of the IgG fraction: fibrin-agar plates (heated and unheated); release of [ 125I]-peptides from plasminogen-free fibrinogen, and the hydrolysis of N-p-tosyl- l-arginine methyl ester. Antibodies to rat tumor fibrinolysin cross-reacted with fibrinolysin(s) produced by cultured human melanoma and breast carcinoma cells, to some preparations of urokinase-activated human plasmin, but had no effect on purified horse and calf plasmin. Rabbit anti-human plasminogen antibodies cross-reacted with the rat tumor fibrinolysin. Inhibition of human plasmin activity was greatly reduced after absorption of goat anti-rat tumor fibrinolysin IgG fraction with human plasminogen, but the fraction remained completely inhibitory for the rat tumor fibrinolysin. Thus, the fibrinolysin secreted by the rat tumor cells was immunologically distinct from human, horse, and calf plasmin.