The coupled photometric assay for plasminogen activator reported by Coleman and Green (1981) Methods in Enzymology (Lorand, L., Ed.), Vol. 80, pp. 408-414, Academic Press, San Diego, CA) has been adapted for use with 96-well plates and an automatic microplates spectrophotometer. The assay allows the discrimination between tissue-type and urokinase-type plasminogen activators in cell culture-conditioned media. It provides a level of detection of these enzymes in the range 10(-17) to 10(-13) mol (determined using purified human plasminogen activators), uses no radioisotopes, and is faster and more economical than similar assays using specific peptide substrates for plasminogen activators. Levels of free plasminogen activator inhibitor activity can be simultaneously measured on the same samples by a simple adaptation of the assay. This method allows an easy treatment of the data by interfacing with a computer and should thus be useful when large numbers of samples are assayed.