The binding of human urokinase-type plasminogen activator (u-PA) to the surface of the human monocytic cell line U937 was studied by immunological detection of bound u-PA or binding of biotinylated diisopropyl fluorophosphate-inactivated human u-PA visualized by light or electron microscopy. Untreated U937 cells showed a characteristic binding pattern, with the majority of the u-PA bound to the microvillar-containing protruding pole of the cells. After treatment with the phorbol ester PMA, the resulting adherent cell population was very heterogeneous with respect to both cellular morphology and u-PA binding. The bound u-PA was distributed on both the dorsal and the substrate side of the cells, and the patches of bound u-PA could not be correlated to any typical membrane conformations or cell-cell or cell-substratum contacts. When a monoclonal antibody directed against the amino-terminal fragment (ATF) of u-PA was used, the results were identical regardless of whether intact u-PA or ATF was used for binding to the cells. In contrast, when a monoclonal antibody recognizing the non-receptor-binding protease domain of u-PA was used, bound ATF showed no staining, while bound intact u-PA was stained as efficiently as above. The alteration of u-PA receptor distribution following treatment with PMA could be related to the changes in glycosylation and ligand affinity of the purified u-PA receptor previously described following PMA treatment of U937 cells.