The his-rfb deletion series of Salmonella typhimurium mutants characterized previously by Nikaido et al. was examined for the presence of the enterobacterial common antigen (ECA). All deletions not extending further to the left than the genes for cytidine phosphoabequose synthesis were ECA positive, whereas longer deletions (extending to the genes for thymidine diphosphorhamnose synthesis or further) were ECA negative. When these long-his-rfb deletion strains were studied further, it became clear that they (four out of four studied) had accumulated a second mutation, called rff, close to ilv, which prevented the synthesis of ECA. When rff- was replaced by rff+, the recombinants, now having the his-rfb deletion only, produced traces of ECA, showed reduced viability, increased sensitivity to sodium dodecyl sulfate (SDS) and to a lesser extent, to other anionic detergents, and accumulated secondary "suppressor" mutations upon storage. Such suppressor-containing mutants could be isolated by selecting for resistance to 1% SDS. Thirty of 46 SDS-resistant mutants studied had a second mutation, which alone prevented the synthesis of ECA, close to ilv. This ilv-linked mutation was similar to the rff mutation of the strains studied originally. The new rff mutation was similar to previously described rfe mutations in its close linkage to ilv and association with an ECA-negative phenotype. It differed from rfe, however, by not affecting the synthesis of the O antigens (O-6,7) of group C1. In Salmonella group C1, all ECA genes identified thus far are linked to ilv (rfe and/or rff) and none is linked to rfb.