Mutation frequency decline (MFD) is the rapid decrease in the frequency of certain induced nonsense suppressor mutations occurring when protein synthesis is transiently inhibited immediately after irradiation. MFD is abolished by mutations in the uvrA, -B, or -C genes, which prevent excision repair, or by a mfd mutation, which reduces the rate of excision but does not affect survival. Using an in vitro repair synthesis assay we found that although wild-type cells repair the transcribed (template) strand preferentially, mfd- cells are incapable of strand-specific repair. The deficiency in strand-selective repair of mfd- cell extract was corrected by adding highly purified "transcription-repair coupling factor" to the reaction mixture. We conclude that mfd is, most likely, the gene encoding the transcription-repair coupling factor.