The formation of cyclopropane fatty acid (CFA) and its role in the acid shock response in Salmonella enterica serovar Typhimurium (S. typhimurium) was investigated. Data obtained by GC/MS demonstrated that the CFA level in S. typhimurium increased upon its entry to the stationary phase, as in other bacteria. The cfa gene encoding CFA synthase was cloned, and mutants of the cfa gene were constructed by allelic exchange. A cfa mutant could not produce CFA and was sensitive to low pH. Introduction of a functional cfa gene into a cfa mutant cell made the mutant convert all unsaturated fatty acids to CFAs and partially restored resistance to low pH. Interestingly, the alternative sigma factor RpoS, which was induced during the stationary phase, affected the production of C(19) CFA but not C(17) CFA. Western blotting analysis showed that the increase in expression of CFA synthase at early stationary phase was due to the alternative sigma factor RpoS.