Analysis of the sequence of a 4.1-kb rfa region downstream from rfaP revealed four genes. The first of these encodes a basic protein of 36,730 Da and does not correspond to any known rfa gene. It has been designated rfaS. The second gene was identified as rfaB on the basis of its ability to complement a Salmonella typhimurium rfaB mutant and encodes a 42,060-Da protein. The third and fourth genes encode proteins of 39,423 and 36,046 Da which are strongly homologous to the RfaI and RfaJ proteins of S. typhimurium. Escherichia coli K-12 restriction fragments carrying these genes complement an S. typhimurium rfaI mutant and, at lower efficiency, an rfaJ mutant. The difference in complementation efficiency suggests that the rfaI and rfaJ genes of E. coli K-12 have sugar and acceptor specificities different from those of S. typhimurium, as predicted from the different lipopolysaccharide (LPS) core structures of the two organisms. Defined mutations affecting all four genes were constructed in vitro and crossed onto the chromosome. The phenotypes of these mutations suggest that extension of the core may require protein-protein interactions between the enzymes involved in core completion as well as the interaction of these enzymes with their specific acceptor molecules. Mutants blocked at rfaI or genes encoding earlier steps in core biosynthesis exhibited a single predominant LPS band on gels while mutants blocked at rfaJ or genes encoding later steps produced multiple strong bands, indicating that one of the processes generating core heterogeneity requires a functional rfaI gene.