Phosphatidic acid (PA) is known to be a crucial phospholipid intermediate in cell membrane biosynthesis. In Escherichia coli, this molecule is produced from lysophosphatidic acid (LPA) by LPA acyltransferase (EC 18.104.22.168), encoded by plsC. E. coli possesses only one such LPA acyltransferase and a plsC mutant is non-permissive for growth at elevated temperatures. This study describes the identification and characterization of two genes from Pseudomonas fluorescens F113 that encode enzymes with LPA acyltransferase activity. One of the genes, hdtS, was previously described, whereas patB is a novel gene. In addition, a putative lyso-ornithine lipid acyltransferase was also identified. All three proteins possess conserved acyltransferase domains and are homologous to PlsC and to LPA acyltransferases identified in Neisseria meningitidis. Functional analysis determined that both HdtS and PatB are functional LPA acyltransferases, as both complemented an E. coli plsC mutant. Mutants lacking each of the putative acyltransferases were constructed and analysed. Growth defects were observed for hdtS and patB single mutants, and a double hdtSpatB mutant could not be constructed. To determine precise roles in phospholipid synthesis, fatty acid methyl ester analysis was carried out. The hdtS mutant displayed a profile consistent with a defect in LPA acyltransferase activity, whereas no such phenotype was observed in the patB mutant, indicating that hdtS encodes the primary LPA acyltransferase in the cell. The presence of at least two genes specifying LPA acyltransferase activity may have implications for the function and survival of P. fluorescens in diverse environments.