The extracellular matrix protein osteopontin (OPN) interacts with a number of integrins, namely αvβ1, αvβ3, αvβ5, α9β1, α8β1, and α4β1. We have investigated the interaction of α5β1 integrin with OPN using K562 cells, which only express α5β1. α5β1 is in a low activation state in this cell line, but can be stimulated to a higher activation state by the phorbol ester TPA. Treating K562 wild-type cells (K562-WT) with TPA stimulated an interaction between α5β1 and OPN. No interaction was seen in the absence of TPA. α5β1 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 α4 integrin in K562 cells (K562-α4β1) stimulated α5β1-dependent binding to aa17–168 in the absence of TPA, suggesting that α4β1 activates α5β1 in K562 cells. Adhesion via α5β1 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 α5β1 integrin can interact with thrombin-cleaved osteopontin when in a high activation state.