The cellular receptor for urokinase-type plasminogen activator (uPAR) is a glycolipid-anchored membrane protein thought to play a primary role in the generation of pericellular proteolytic activity, and to be involved in cancer cell invasion and metastasis. This protein is composed of three homologous domains, the NH2-terminal of which is involved in the high-affinity binding (Kd approximately 0.1-1.0 nM) to the epidermal growth factor-like module of urokinase-type plasminogen activator (uPA). Here we report that intact uPAR binds the low molecular weight fluorophore 8-anilino-1-naphthalenesulfonate (ANS) to form a 1:1 stoichiometric complex and that the resulting enhancement of the ANS fluorescence probes the functional state of uPAR as judged by several independent criteria. First, the uPAR-mediated increase in ANS fluorescence can be titrated by uPA as well as by its receptor binding derivatives (the amino-terminal fragment and the growth factor-like module). Second, an anti-uPAR monoclonal antibody, capable of preventing uPA binding, can also titrate the uPAR-dependent ANS fluorescence whereas other antibodies not interfering with uPA binding are unable to exert this effect. Third, the dissociation profile of uPA-uPAR complexes as a function of increasing concentrations of guanidine hydrochloride closely parallels the loss of the ANS binding site in uPAR. Finally, liberation of the NH2-terminal domain from uPAR by limited chymotrypsin cleavage after Tyr87 leads to a loss of both enhanced ANS fluorescence and high-affinity uPA binding.(ABSTRACT TRUNCATED AT 250 WORDS)