Peptides from different regions of the poliovirus type 1 capsid protein VP1 were synthesized. Antibodies raised against these peptides in rabbits and rats recognized the cognate peptides and denatured VP1. Peptides from four regions of VP1 generated antisera with neutralizing titers specifically against poliovirus type 1. Antisera against all other regions of VP1 failed to neutralize virus infectivity, although some of the antisera clearly bound to native virions. Thus, the neutralizing determinants on VP1 reside in specific noncontiguous regions of the protein and can be defined by specific peptides from these regions.