Reovirus infection is initiated by interactions between the attachment protein sigma1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein lambda2, which serves as the structural base for sigma1, suggests that integrins mediate reovirus endocytosis. A beta1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a beta1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in beta1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing beta1. However, reovirus bound equivalently to cells that differed in levels of beta1 expression, suggesting that beta1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into beta1-deficient cells was substantially diminished in comparison to viral uptake into beta1-expressing cells. These data provide evidence that beta1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.