Gram-positive bacteria, including Listeria monocytogenes, adjust membrane fluidity by shortening the fatty acid chain length and increasing the proportional production of anteiso fatty acids at lower growth temperatures. The first condensation reaction in fatty acid biosynthesis is carried out by beta-ketoacyl-acyl carrier protein synthase III (FabH), which determines the type of fatty acid produced in bacteria. Here, we measured the initial rates of FabH-catalyzed condensation of malonyl-acyl carrier protein and alternate branched-chain precursor acyl-CoAs utilizing affinity-purified His-tagged L. monocytogenes FabH heterologously expressed in Escherichia coli. Listeria monocytogenes FabH showed a preference for 2-methylbutyryl-CoA, the precursor of odd-numbered anteiso fatty acids, at 30 degrees C, which was further increased at a low temperature (10 degrees C), suggesting that temperature-dependent substrate selectivity of FabH underlies the increased formation of anteiso branched-chain fatty acids during low-temperature adaptation. The increased FabH preferential condensation of 2-methylbutyryl-CoA could not be attributed to a significantly higher availability of this fatty acid precursor as acyl-CoA pool levels were reduced similarly for all fatty acid precursors at low temperatures.