A simple specific and reproducible method for determination of plasminogen activator activity in rat ovaries has been developed by using the chromogenic substrate S-2251. The two steps of enzymatic reactions, i.e. activation of plasminogen and subsequent hydrolysis of the substrate was performed in one step incubation. A linear relationship was observed between the amount of chromogen produced and activator activity in the range of the optical density form 0.05 to 1.20 for 30 min's incubation. Endogenous activity of non-specific proteases, plasmin or plasmin inhibitors which might be contained in rat ovaries turned out not to interfere with the specificity of a standardized assay procedure. Reproducibility was firmly established with coefficient of variation not exceeding 10%. Using this method, a marked increase followed by a drastic decrease in the activator activity was shown with rat ovaries around the time of ovulation after the injection of human chorionic gonadotropin.