Distinct binding interactions between cell-surface receptors and extracellular matrix components are characteristic of multifunctional adhesion proteins such as vitronectin. The close proximity of binding sites for αv-integrins and plasminogen activator inhibitor-1 (PAI-1) on vitronectin may have consequences for cell adhesion and migration, or for the localized inhibition of plasminogen activators. In this study, the kinetics and reversibility of vitronectin-dependent cell adhesion via αv-integrins was investigated using RGD peptides and PAI-1 as competitors. Active, but not latent or cleaved PAI-1, and RGD peptides were effective in preventing cell adhesion to vitronectin provided the inhibitor was present at the time of cell seeding. In a concentration-dependent manner urokinase or thrombin abrogated the inhibitory effect of PAI-1. Following cell seeding onto a vitronectin substratum, delayed addition of RGD peptides or active PAI-1 (10−20 min post-seeding) resulted in the loss of their inhibitory potential. These data were supported by experiments in a purified system where delayed addition of active PAI-1 could no longer prevent vitronectin binding to immobilized αvβ3, while a cyclic RGD peptide gave some moderate inhibition. The apparent stabilization of vitronectin−integrin contacts was observed with immobilized native or multimeric vitronectin but not with the more rigid form of denatured, aggregated multimers. These results demonstrate that the cell adhesive properties of vitronectin depend on its conformational flexibility and can be tightly regulated in a spatio-temporal manner through direct competition of cellular integrins by soluble or matrix-bound factors such as PAI-1.