The recognition sites for human antibodies which are cross-reactive between different types of enteroviruses were determined and characterized. Serum samples obtained from 58 patients with culture-confirmed enteroviral infections were analyzed in enzyme immunoassays against two sets of overlapping synthetic peptides covering residues 31 to 96 of poliovirus 1 VP1 (Mahoney strain) and residues 31 to 148 of coxsackievirus B1 VP1 (position based on alignment with poliovirus 1 VP1, Mahoney strain). A major antigenic region eliciting cross-reactive antibodies could be located to residues 37 to 51 of VP1. Furthermore, a single peptide covering residues 42 to 55 almost completely inhibited the binding of human antibodies to heat-inactivated enteroviruses, indicating that residues 42 to 55 of VP1 contain a major region eliciting cross-reactive antibodies. By using peptide analogs in which each residue within positions 42 to 55 of VP1 was sequentially substituted by Ala or Gly, we were able to determine the most essential residues for human antibody binding in 38 of the convalescent-phase patient serum samples. In a majority of the serum samples, the most essential residues for antibody binding were found to be Pro-42, Ala-43, Leu-44, Thr-45, Ala-46, Glu-48, Thr-49, and Gly-50. All of these residues are conserved, according to known enterovirus sequences, with the divergent echovirus 22 excepted. In conclusion, we could demonstrate that the essential residues for binding of cross-reactive antibodies are well conserved within the enterovirus family. These findings provide a molecular basis for the observed antibody cross-reactivity within the enterovirus group.