Mutants resistant to the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofurano-sylbenzimidazole (DRB) have been isolated in the Chinese hamster ovary cell line CHO-K1. Three independently isolated mutants, DRB6 DRB10, and DRB13, were 3-, 5-, and 3.5-fold, respectively, more resistant to DRB than the parental cell line WTCHO. The DRB-resistant mutations were expressed codominantly in somatic cell hybrids of DRB-resistant and DRB-sensitive cell lines. In vivo treatment of CHO-K1 cells with DRB resulted in specific inhibition of endogenous RNA polymerase II activity in cell lysates. Whereas DRB inhibited RNA polymerase II activity in WTCHO cells by a maximum of 60% at concentrations as low as 60 microM, 300 microM DRB was required to inhibit 60% of the RNA polymerase II activity in DRB10 cells. However, the inhibition of the DRB-sensitive RNA polymerase II activity in DRB10 was biphasic. About half (53 to 56%) of this activity was inhibited by 90 microM DRB and thus showed a DRB sensitivity similar to the wild-type RNA polymerase II activity; the remaining DRB-sensitive RNA polymerase II activity was maximally inhibited by 300 microM DRB. These results indicated that there were two copies of the drbR locus (drb+ and drbR-10) in DRB10 and confirmed that the drbR-10 mutation was expressed codominantly. Somatic cell hybrids of DRB-resistant and alpha-amanitin-resistant cell lines grew in medium containing both DRB and alpha-amanitin, demonstrating that the drbR and amaR mutations were not in the same gene. Thus, the drbR mutations may define an additional component of the RNA polymerase II transcriptional complex in mammalian cells.