The mouse-adapted strain of poliovirus type 2 (Lansing) induces fatal poliomyelitis in mice after intracerebral inoculation, whereas mice inoculated with poliovirus type 1 (Mahoney) show no signs of disease. Previous work indicated that the adaptation to mouse virulence is associated with the viral capsid proteins and that mutations in neutralization antigenic site I of poliovirus reduce neurovirulence of the Lansing strain in mice. The role of antigenic site I in mouse neurovirulence was further explored by constructing an antigenic hybrid virus. Six amino acids in antigenic site I of the Mahoney strain were replaced with a sequence specific for the Lansing strain by using a mutagenesis cartridge. The hybrid virus was neutralized by polyclonal antisera elicited by the type 1 and type 2 strains of poliovirus and by neutralizing monoclonal antibodies directed against antigenic site I of type 2 virus. The hybrid virus induced paralytic disease in mice, an observation demonstrating that a short sequence of amino acids in antigenic site I is an important determinant of poliovirus host range. Antigenic site I may be involved in attachment of poliovirus to cells of the mouse central nervous system.