In this study we describe BI-K0058, a new inhibitor of the transcription-termination factor Rho belonging to a different chemical class from bicyclomycin, the only known antibiotic acting on Rho. BI-K0058 inhibits the poly(C)-dependent ATPase activity of Rho with an IC(50) of 25 microM as well as in vitro transcription-termination of two natural substrates, the Salmonella enterica hisG cistron and the f1 phage intergenic region. BI-K0058 does not affect photolabeling of Rho by ATP. The results of gel mobility shift experiments with a natural RNA substrate demonstrate that BI-K0058 inhibits the formation of the ATP-independent high affinity Rho-RNA complex.