Plasminogen activators (PA), particularly the lower Mr urokinase (u-PA) type, have been associated with tumor cell invasion and metastasis. We have examined the expression of PA by two human prostate cancer cell lines (PC-3 and DU-145) using functional and immunologic techniques. The culture media and cell extracts of the more aggressive PC-3 cell line contained more than two-fold greater PA activity than the relatively indolent DU-145 cell line. Zymographic studies identified the PA expressed as u-PA. PC-3 cells expressed an additional lower molecular weight form of u-PA not noted in DU-145 cells. Heterogeneity in u-PA expression was shown by the fibrin lysis assay, immunohistochemistry, and dual parameter flow cytometry indicating the presence of phenotypically divergent cell populations. Increased u-PA expression may identify those tumor cells that possess aggressive biological potential.