Analysis of the nucleotide sequence of the rfbX gene of Shigella flexneri revealed that it contained a high proportion of rare codons, as previously observed in the analysis of the O-antigen polymerase-encoding gene rfc [Morona et al., J. Bacteriol. 176 (1994) 733-747]. The rfbX gene encodes a hydrophobic, 46-kDa protein, with 12 potential transmembrane-spanning domains, that shows structural homology with gene products encoded in many rfb regions, and with Orf0416 of the rff region of Escherichia coli K-12 which has also been identified as a member of this class of proteins. Attempts to clone rfbX independent of other rfb genes, and to identify the protein product of rfbX have proven unsuccessful. Analysis of plasmids containing various deletions within the rfb region suggest that the 5' end of rfbX plays an indirect regulatory role in expression of the dTDP-rhamnose biosynthetic enzymes, encoded by rfbBCAD. We speculate that RfbX is a cytoplasmic membrane protein which functions in the transport of the O-antigen repeat unit.