The peptide chain release factor 2 (RF2) gene, prfB, was cloned from Salmonella typhimurium by DNA hybridization using the Escherichia coli prfB probe. The nucleotide and amino acid sequences of prfB are 87.0% and 95.6% homologous between E. coli and S. typhimurium, respectively, including an in-frame premature UGA stop codon at position 26, the site of +1 frameshift for mature RF2 synthesis. The supK584 mutation, which had been isolated as a recessive UGA suppressor in S. typhimurium, caused an opal (UGA) substitution at amino acid position 144 in the prfB gene. Complementation, reversion, and gene fusion analyses led to the conclusion that supK is a S. typhimurium RF2 mutation and this opal RF2 mutation generates a UGA suppressor activity, presumably because of inefficient translation termination due to the reduced cellular level of RF2. In fact, suppression of the supK opal mutation results from a form of autogenous control of RF2 synthesis.