Sequence analysis of poliovirus vaccine RNA has resulted in the identification of a new point mutation at position 2493 in Sabin 3. cDNA-derived Sabin 3 virus whose genome includes the new mutation has phenotypic properties expected for a vaccine strain. Virus engineered to contain a single base substitution at 2493 has lost the small plaque phenotype, and low neurovirulence characteristics normally associated with Sabin poliovirus strains. The significance of these observations and a potential relationship to the frequency of vaccine-associated poliomyelitis are described.