Radiolabeled pro-urokinase plasminogen activator (pro-uPA) was cross-linked to a specific protein on the surface of human monocyte-like U937 cells in a reaction catalyzed by tissue transglutaminase. The conjugate formed with this unknown component had a much higher molecular weight (apparent M(r) 250,000-300,000) than the complex of pro-uPA and the urokinase plasminogen activator receptor (uPAR). There was a strong preference for the pro-form of uPA. The conjugate was recognized by antibodies against uPA but not by anti-uPAR antibodies. Nevertheless, the blocking of uPAR with a monoclonal antibody abolished the formation of the conjugate, thus showing a role of uPAR in this process.