Rotavirus NSP4 is a viral enterotoxin capable of causing diarrhea in neonatal mice. This process is initiated by the binding of extracellular NSP4 to target molecule(s) on the cell surface that triggers a signaling cascade leading to diarrhea. We now report that the integrins alpha1beta1 and alpha2beta1 are receptors for NSP4. NSP4 specifically binds to the alpha1 and alpha2 I domains with apparent K(d) = 1-2.7 muM. Binding is mediated by the I domain metal ion-dependent adhesion site motif, requires Mg(2+) or Mn(2+), is abolished with EDTA, and an NSP4 point mutant, E(120)A, fails to bind alpha2 integrin I domain. NSP4 has two distinct integrin interaction domains. NSP4 amino acids 114-130 are essential for binding to the I domain, and NSP4 peptide 114-135 blocks binding of the natural ligand, collagen I, to integrin alpha2. NSP4 amino acids 131-140 are not associated with the initial binding to the I domain, but elicit signaling that leads to the spreading of attached C2C12-alpha2 cells, mouse myoblast cells stably expressing the human alpha2 integrin. NSP4 colocalizes with integrin alpha2 on the basolateral surface of rotavirus-infected polarized intestinal epithelial (Caco-2) cells as well as surrounding noninfected cells. NSP4 mutants that fail to bind or signal through integrin alpha2 were attenuated in diarrhea induction in neonatal mice. These results indicate that NSP4 interaction with integrin alpha1 and alpha2 is an important component of enterotoxin function and rotavirus pathogenesis, further distinguishing this viral virulence factor from other microbial enterotoxins.