The human integrin very late antigen (VLA)-2 (CD49b/CD29) mediates interactions with collagen and is the receptor for echovirus 1. Binding sites for both collagen and echovirus 1 have been mapped to the I domain within the alpha2 subunit of the VLA-2 alpha2beta1 heterodimer. Although murine VLA-2 interacts with collagen, it does not bind virus. We have used isolated human-murine chimeric I domains expressed as glutathione S-transferase fusion proteins in Escherichia coli to identify two groups of amino acids, 199-201 and 212-216, independently involved in virus attachment. These residues are distinct from the metal ion-dependent adhesion site previously demonstrated to be essential for VLA-2 interactions with collagen. Mutations in three metal ion-dependent adhesion site residues that abolish adhesion to collagen had no effect on virus binding. These results confirm that different sites within the I domain are responsible for VLA-2 interaction with extracellular matrix proteins and with viral ligands.