We have previously demonstrated that the N-terminal immunoglobulin-like domain (domain 1; 115 amino acids) of human poliovirus receptor (hPVR) is essential for poliovirus binding and infection to cells. To identify amino acids involved in the interaction with poliovirus, we constructed a number of cDNAs encoding mutant hPVRs whose domain 1 was partially derived from mouse PVR (mPVR) homolog, which does not serve as a binding site for poliovirus. Poliovirus binding and infection assays were performed on mouse L cells that express these chimera cDNAs. Anti-hPVR monoclonal antibodies were employed to confirm the presence of mutant PVRs on the surface of mouse cells and to know conformational alteration of these PVRs. A significant decrease in efficiency of both poliovirus binding and infection to the cells was observed when one or a few amino acids of hPVR at Gly73, Ser74, Gln82, Leu99-Glu102, or Gln130-Ser132 were substituted by the corresponding amino acids of mPVR. Similar results were obtained when a 2-amino acid insertion of mPVR, which was missing in hPVR, was introduced at the corresponding site (between Arg98 and Leu99) of hPVR. These amino acids were highly conserved in functional PVRs of primates but not in unfunctional PVRs of rodents. These results indicate that the amino acids identified may have important roles in interaction of PVR with poliovirus that leads to the establishment of the virus infection. In the three-dimensional model of the domain 1 of hPVR, these amino acids are located on one side of the molecule. This suggests that the interaction with poliovirus occurs on this side of the domain 1.