We examined the phospholipids (Phls) and the membrane fatty acid (FA) composition in Salmonella enterica serovar Typhimurium dam and/or seqA mutants. Phosphatidylglycerol, phosphatidylethanolamine (PE), and cardiolipin (CL) are the major Phls present in all the strains and accounted for greater than 95% of the total lipid phosphorus. Phosphatidic acid and phosphatidylserine are the minor ones. The seqA mutant showed a decrease in PE and an increase in CL and phosphatidylglycerol proportion compared with the wild-type strain. The same changes were observed with the seqA dam double mutant. However, the dam mutation caused an unusual accumulation of CL with a significant decrease in the PE content, compared with the isogenic wild-type strain. FA composition of the total lipids and the different fractions containing Phls have been determined. The major saturated FAs (SFAs) and unsaturated FAs (UFAs) found were C(14:0), C(16:0) and C(16:1w7), C(18:1w9), respectively. Cyclic FAs, cyc(17:0) and cyc(19:0), were also present in appreciable amounts. Moreover, dam and/or seqA mutations caused a decrease in UFA/SFA ratio and there was a progressive reduction in the content of C(16:1w7) and C(18:1w9), going through the order seqA, dam/seqA, and dam mutants. This decrease in UFA content was compensated for in all strains by an increase in the corresponding C(17-) and C(19-) cyclic FAs. So these UFAs were converted to their cyclopropane derivatives, which resulted in a low UFA/SFA ratio. SeqA and Dam proteins might regulate FA biosynthesis and Phls composition of Salmonella enterica serovar Typhimurium.