The cellular receptor for urokinase plasminogen activator (uPA-R) is a monomeric phosphatidylinositol-linked glycoprotein (gp40-65) that may contribute to the invasive capacity of tumor and inflammatory cells by focusing the activity of urokinase (uPA) in converting plasminogen to plasmin, a serine protease capable of degrading extracellular matrix proteins. The further characterization of uPA-R has been facilitated by our recent development of a monoclonal antibody, anti-Mo3f, specific for uPA-R. This mAb bound to uPA-R expressed by phorbol myristate acetate-stimulated U-937 cells and by NIH-3T3 cells permanently transfected with uPA-R cDNA. In competitive binding assays, anti-Mo3f inhibited the binding of fluorescein-conjugated uPA ligand to uPA-R expressed by U-937 cells and uPA-R transfectants; conversely, preexposure of cells to saturating quantities of exogenous uPA partially blocked the subsequent binding of anti-Mo3f mAb to uPA-R. Anti-Mo3f mAb was employed as the capture reagent in an ELISA for the quantitation of soluble forms of uPA-R (derived from U-937 cells and recombinant uPA-R) which had a sensitivity of approximately 4-12 ng/ml. Anti-Mo3f mAb was also applied as a serologic probe for the detection of uPA-R expressed by human tumor tissues. By immunoperoxidase staining, anti-Mo3f demonstrated positive tumor cell staining in 4 of 16 breast and 7 of 31 prostate carcinomas in formalin-fixed, paraffin-embedded specimens. These data indicate that the anti-Mo3f mAb detects an epitope proximate to or within the ligand binding domain (domain 1) of uPA-R and may be useful as a tool for the serologic detection of uPA-R in soluble form or associated with human tumors.