Cyclopropane fatty acyl synthases (CFA synthases) are enzymes that catalyse the addition of a methylene group across cis double bonds of monounsaturated fatty acyl chains in lipids. We have investigated the function of two putative genes, cfa1 and cfa2, proposed to code for CFA synthases in Sinorhizobium meliloti. Total fatty acid composition and fatty acid distributions within lipid classes for wild-type and cfa1 and cfa2 mutant strains grown under Pi starvation and in acidic culture conditions were obtained by GC/MS and by infusion ESI/MS/MS, respectively. For wild-type cells and the cfa1 mutant, total cyclopropane fatty acids (CFAs) increased by 10% and 15% under Pi starvation and acidic conditions, respectively; whereas in the cfa2 mutant, CFAs were less than 0.1% of wild-type under both growth conditions. Reporter gene fusion experiments revealed that cfa1 and cfa2 were expressed at similar levels in free-living cells. Thus under the conditions we examined, cfa2 was required for the cyclopropanation of lipids in S. meliloti whereas the role of cfa1 remains to be determined. Analysis of intact lipids revealed that cyclopropanation occurred on cis-11-octadecenoic acid located in either the sn-1 or the sn-2 position in phospholipids and that cyclopropanation in the sn-2 position occurred to a greater extent in phosphatidylcholines and sulfoquinovosyldiacylglycerols under acidic conditions than under Pi starvation. The cfa2 gene was also required for cyclopropanation of non-phosphorus-containing lipids. Principal components analysis revealed no differences in the cyclopropanation of four lipid classes. We concluded that cyclopropanation occurred independently of the polar head group. Neither cfa1 nor cfa2 was required for symbiotic nitrogen fixation.