Previous studies have shown that the genome of Sabin type 3 poliovaccine strain (P3/Leon 12a₁b) possesses a diminished translation efficiency as compared to genomes of closely related neurovirulent strains, the neurovirulent progenitor (P3/Leon/37), or a revenant (P3/119/70) of the vaccine (Y. V. Svitkin, S. V. Maslova, and V. 1. Agol, 1985, Virology 147, 243–252). Here we attempted to evaluate the contribution of each mutation in the genome of the vaccine to this translation deficiency. Recombinants between P3/Leon 12a₁b and P3/Leon/37 or P3/119/70 were constructed in vitro and their RNAs were translated in a cell-free system derived from Krebs-2 cells. The results show that of 10 nucleotide differences between the genomes of P3/Leon 12a₁b and P3/Leon/37 9 have minor or no effect on translation and that the only mutation of significance is C₄₇₂ → U which is known to reduce the neurovirulence of the virus. Reversion from uridine to cytosine at position 472 in type 3 poliovaccine upon replication in the human gut resulted in an increase of both translation efficiency of polio RNAs and neurovirulence of corresponding strains. The data provide evidence for a common nucleotide sequence regulatory element for protein synthesis of the virus and its neurovirulence. In vitro translation assays may therefore prove to be useful for detection of attenuating mutations in the 5′ noncoding region of poliovirus genome. The apparent involvement of the translation mechanism in the expression of neurovirulent or attenuated phenotype of poliovirus is briefly discussed.