A conditional-lethal rho mutant of Salmonella typhimurium LT2 has been isolated. The mutation was selected as a suppressor of the polarity of an insertion sequence (IS)2-induced mutation (gal3) carried on an F' plasmid. In addition to suppression of IS2-induced polarity, the rho-ill mutation suppressed nonsense and frameshift polarity. The rho-associated polycytidylic acid-dependent adenosine triphosphatase activity in the mutant strain was elevated 15-fold above that in the parental strain, and the mutant rho protein was thermally unstable. A temperature-resistant revertant of the mutant strain did not suppress polarity and contained normal levels of polycytidylic acid-dependent adenosine triphosphatase, suggesting that the phenotype of the rho-ill-bearing strain is the consequence of a single mutation. The rho-ill mutation was located on the S. typhimurium linkage map midway between the ilv and cya loci by phage P22 cotransduction studies. F' plasmid maintenance was not impaired in the mutant strain, and the mutation was recessive to the wild-type allele. The rho-ill mutation did not alter in vivo expression of either the tryptophan or histidine operons. This study; see text B. Ames Wild-type; B. Ames R. 0. Burns (5) J. Roth (15) J. Roth e Genetic symbols are those of Sanderson and Hartman (33). The his operator, formerly hisO, is written hi8Go. b (P1) or (P22) indicates source of transducing lysate. ' Nomenclature conventions for insertion mutations follow the suggestions of Chumley et al. (8).