The expression of virulence factors such as hemolysin and lipopolysaccharides in Proteobacteria is regulated by the transcription elongation factor RfaH. RfaH reduces pausing and termination at intergenic sites, and thus allows RNA polymerase to conclude transcription of the distal genes in long virulence operons. The yaeQ gene of Salmonella enterica sv. Typhimurium has been identified as a high-copy-number suppressor of the hemolytic defect in an rfaH deletion strain, leading to speculation regarding a direct role of YaeQ in the transcriptional control of bacterial virulence. In order to evaluate this hypothesis, yaeQ genes from Escherichia coli and S. enterica sv. Typhimurium were cloned and expressed. Their products, purified YaeQ proteins, displayed no antitermination effects in in-vitro transcription assays over a wide range of concentrations, neither by themselves nor in competition with RfaH. When overexpressed in vivo, plasmid-borne E. coli and S. enterica sv. Typhimurium yaeQ genes also failed to restore hemolytic activity in an rfaH deletion strain under conditions in which episomal E. coli rfaH and its orthologs exhibited full complementation of the genomic rfaH deletion. Taken together, our findings do not support the hypothesis of YaeQ involvement in RfaH-dependent regulation of virulence, even in stoichiometric excess in vitro or upon overexpression in vivo.