In the present paper we have analyzed uPAR-mediated cellular binding to vitronectin using the murine erythroid progenitor cell line 32D. We show that expression of uPAR in 32D cells promotes cellular binding to vitronectin, but fails to support cell spreading. The strength of binding is correlated to the expression level of uPAR and is strongly stimulated by the presence of uPAR ligands. Using a truncated variant of uPAR lacking domain 1 and by antibody inhibition experiments, we demonstrate that domain 1 plays a crucial role in uPAR-mediated cellular binding. The failure of the mutant uPAR to promote cellular binding is paralleled by a strong reduction in the affinity for vitronectin in vitro.