The antifibrinolytic activity of cytosol from Dunning R3327H rat prostate tumors was studied. The prostate tumors from rats treated with D-Trp6-LH-RH had 2.5 times lower plasminogen activator activity than tumors from untreated rats. This was due to the presence of an inhibitor of plasminogen activator as well as a reduction in residual activity of plasminogen activator(s). Only the cytosolic extracts from prostate tumors of rats treated with D-Trp6-LH-RH contained this inhibitor. The purified inhibitor (m.w. 21,000), formed a complex with urokinase and partially purified plasminogen activator(s) from prostate tumors of untreated as well as D-Trp6-LH-RH treated rats. The increase in antifibrinolytic activity after treatment with LH-RH analogs may be an important factor in reducing the invasiveness of the prostate tumor.