Antigenic analysis of coxsackievirus A9 (CAV9) was carried out by using a peptide scanning method. Immunogenic regions in the capsid proteins VP1, VP2, and VP3 were recognized by antibodies in the sera of virus-immunized rabbits. The peptide sequences were scanned using a 12-amino-acid window and three-residue shift. Three immunogenic regions, located in the N- and C-terminal parts of VP1 and in the N-terminus of VP3, were identified. Trypsin treatment of the virus, known to cleave off the C-terminus of VP1 containing a functional RGD motif, completely abolished the reactivity against this region but did not have any other significant effect on antigenicity. In further studies, it was found that the RGD motif itself was poorly immunogenic whereas antibody-binding sites were located at both sides of the motif. New antigenic sites emerged after heat treatment of CAV9 at 56 or 100 degrees C prior to immunization; in particular, loop structures between beta strands in VP2 exhibited increased immunogenicity. New antigenic sites in VP1 and VP3 also appeared after the treatments. In spite of the markedly altered reactivity in peptide scanning, the virus treated at 56 degrees C elicited high titers of neutralizing antibodies. To reveal cross-reactive antigenic sites, antisera raised against coxsackievirus B3 and echovirus 11 were also tested. The cross-reactive antigenic sites were located mainly in the N-terminal parts of VP1 and VP3.