Human parechoviruses 1 and 2 (HPEV1 and HPEV2, respectively), formerly known as echoviruses 22 and 23, have been assigned to a novel picornavirus genus on the basis of their distinct molecular and biological properties. To study the immunological characteristics of HPEV1 capsid proteins, antigenic analysis was carried out by a peptide scanning technique, which can be used to identify the immunogenic peptide sequences of a protein. Partially overlapping peptides, representing the capsid of HPEV1, were synthesized using a 12 aa window in a three residue shift and reactivity of rabbit and murine HPEV1 antisera against these peptides were tested. Using this method, an antigenic site in the VP0 polypeptide, recognized by both rabbit and murine antisera, was identified. The sequence of this region was conserved among HPEV1 clinical isolates obtained from Finland and the United States. Antiserum against this peptide region showed neutralizing activity against HPEV1 in cell culture. Because the C-terminal region of HPEV1 VP1 contains a functional RGD motif, the antigenicity of this region was also tested. By using the corresponding peptide antiserum, neutralization of HPEV1 was observed. Cross-neutralization between HPEV1 and coxsackievirus A9, an enterovirus with a similar RGD motif in VP1, was also detected.