Rotavirus contains two outer capsid viral proteins, the spike protein VP4 and major capsid component VP7, both of which are implicated in cell entry. We show that VP4 and VP7 contain tripeptide sequences previously shown to act as recognition sites for integrins in extracellular matrix proteins. VP4 contains the alpha2beta1 integrin ligand site DGE. In VP7, the alphaxbeta2 integrin ligand site GPR and the alpha4beta1 integrin ligand site LDV are embedded in a novel disintegrin-like domain that also shows sequence similarity to fibronectin and the tie receptor tyrosine kinase. Microorganism sequence homology to these ligand motifs and to disintegrins has not been reported previously. In our experiments, peptides including these rotaviral tripeptides and mAbs directed to these integrins specifically blocked rotavirus infection of cells shown to express alpha2beta1 and beta2 integrins. Rotavirus VP4-mediated cell entry may involve the alpha2beta1 integrin, whereas VP7 appears to interact with alphaxbeta2 and alpha4beta1 integrins.