The extracellular matrix protein osteopontin (OPN) interacts with a number of integrins, namely alpha v alpha 1, alpha v beta 3, alpha v beta 5, alpha 9 beta 1, alpha 8 beta 1, and alpha 4 beta 1. We have investigated the interaction of alpha 5 beta 1 integrin with OPN using K562 cells, which only express alpha 5 beta 1. alpha 5 beta 1 is in a low activation state in this cell line, but can be stimulated to a higher activation state by the phorbol ester TPA TreatingK562 wild-type cells (K562-WT) with TPA stimulated an interaction between alpha 5 beta 1 and OPN. No interaction was seen in the absence of TPA. alpha 5 beta 1 selectively interacted with a GST fusion protein of the N-terminal fragment of OPN (aa17-168), which is generated in vivo by thrombin cleavage of OPN. Expression of the alpha 4 integrin in K562 cells (K562-alpha 4 beta 1) stimulated alpha 5 beta 1-dependent binding to aa17-168 in the absence of TPA, suggesting that alpha 4 beta 1 activates alpha 5 beta 1 in K562 cells. Adhesion via alpha 5 beta 1 is mediated by the Arg-Gly-Asp (RGD) motif of OPN, as mutating this sequence to Arg-Ala-Asp (RAD) blocked binding of both cell types. These data demonstrate that thrombin cleavage regulates the adhesive properties of OPN and that alpha 5 beta 1 integrin can interact with thrombin-cleaved osteopontin when in a high activation state.