Cyclopropane fatty acid (CFA) formation is a post‐synthetic modification of the lipid bilayer that occurs as cultures of Escherichia coli and many other bacteria enter stationary phase. We report the first distinct phenotype for this membrane modification; early stationary phase cultures of strains lacking CFA (as a result of a null mutation in the cfa gene) are abnormally sensitive to killing by a rapid shift from neutral pH to pH 3. This sensitivity to acid shock is dependent on CFA itself because resistance to acid shock is restored to cfa mutant strains by incorporation of CFAs from the growth medium or by introduction of a functional cfa gene on a plasmid. The synthesis of CFA depends in part on the RpoS sigma factor, but the role of RpoS in resistance to acid shock involves additional factors because strains with null mutations in both cfa and rpoS are more sensitive to acid shock than either single mutant strain. Exponential phase cultures of E. coli are much more sensitive to acid shock than stationary phase cultures, but survival is greatly increased if the exponential phase cultures are exposed to moderately acid conditions (pH 5) before shift to pH 3. We show that exposure to moderately acid conditions gives a marked increase in cfa transcription. The efficiency of the survival of acid shock is extremely strain dependent, even among putative wild‐type strains. Much, but not all, of this variability can be explained by the partially or totally defective RpoS alleles carried by many strains.