The enzyme urokinase-type plasminogen activator (uPA) plays a role in cancer invasion, and high levels of uPA in detergent extracts of mammary cancer tissue have been reported to be associated with a poor prognosis. We have explored the possibility of using mammary cancer cytosol extracts routinely prepared for steroid receptor analysis for retrospective prognostic studies of uPA. A sandwich enzyme-linked immunosorbent assay (ELISA) for uPA was developed, using polyclonal catching antibodies and a mixture of three biotinylated monoclonal detecting antibodies, that were selected to recognize free uPA, inhibitor-bound uPA, and uPA bound to its cell surface receptor. The assay detects active uPA and its inactive proenzyme form, pro-uPA, equally well. The limit of detection is approximately 1 pg of pro-uPA in a volume of 100 microliters, and there is a linear dose-response up to 100 pg pro-uPA. The efficiency in extracting uPA of a neutral non-detergent buffer used to prepare cytosol extracts was compared with that of 4 other buffers. There was a pronounced difference in the efficiency, the most efficient being a pH 4.2 buffer containing the non-ionic detergent Triton X-100, while the least efficient was the buffer used to prepare cytosols. Nevertheless, uPA immunoreactivity was readily measurable in the cytosols, and there was a close correlation between the amounts of uPA extracted under optimal conditions and those routinely used for steroid hormone receptor analysis.(ABSTRACT TRUNCATED AT 250 WORDS)