The termination of protein synthesis in bacteria requires two codon-specific polypeptide-release factors, RF-1 and RF-2. A third factor, RF-3, stimulates the RF-1 and RF-2 activities in vitro. To clarify the in vivo role of RF-3 for the RF-2 dependent termination, we isolated and characterized suppressor mutations for the temperature-sensitive RF-2 mutation prfB286. One of the intergenic suppressor mutations, srb-1, acquired an up-promoter alteration in the RF-3 gene, which enhanced the RF-3 expression four- to fivefold. Consistently a threefold increase in the RF-3 level by a promoter-controlled expression plasmid suppressed prfB286. On the other hand, a temperature-sensitive mutation in RF-1, prfA1, was suppressed only slightly by the high-level expression of wild-type RF-3. The RF-3 mutations that suppress prfA1 were isolated and named sra. They were classified into four specific alleles; two each in the N and C-terminal regions. These altered RF-3 proteins restored the RF-1-dependent termination at UAG in prfA1 cells. Moreover, they enhanced the RF-2-dependent UGA termination in both wild-type and prfB286 cells. The termination-stimulating activity of RF-3 was further additively increased by the double sra mutations, suggesting that they affected two distinct protein domains that modulate the termination reaction. Taking these and other results into consideration, RF-3 is likely to interact functionally and cooperatively with the release factors RF-1 and RF-2 in Escherichia coli.