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ABSTRACTWe have identified the gene for transcription termination factor Rho in Staphylococcus aureus. Deletion ofrhoin S. aureusreveals that it is not essential for viability or virulence. We also searched the available bacterial genomic sequences for homologs of Rho and found that it is broadly distributed and highly conserved. Exceptions includeStreptococcus pneumoniae, Streptococcus pyogenes, Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum,and Synechocystissp. strain PCC6803, all of which appear not to possess a Rho homolog. Complementation studies indicate that S. aureusRho possesses the same activity as Escherichia coliRho and that the Rho inhibitor bicyclomycin is active against S. aureusRho. Our results explain the lack of activity of bicyclomycin against many gram-positive bacteria and raise the possibility that the essentiality of rhomay be the exception rather than the rule.