In the bacterial type II fatty acid synthase system, beta-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) catalyzes the condensation of acetyl-CoA with malonyl-ACP. We have identified, expressed, and characterized the Streptococcus pneumoniae homologue of Escherichia coli FabH. S. pneumoniae FabH is approximately 41, 39, and 38% identical in amino acid sequence to Bacillus subtilis, E. coli, and Hemophilus influenzae FabH, respectively. The His-Asn-Cys catalytic triad present in other FabH molecules is conserved in S. pneumoniae FabH. The apparent K(m) values for acetyl-CoA and malonyl-ACP were determined to be 40.3 and 18.6 microm, respectively. Purified S. pneumoniae FabH preferentially utilized straight short-chain CoA primers. Similar to E. coli FabH, S. pneumoniae FabH was weakly inhibited by thiolactomycin. In contrast, inhibition of S. pneumoniae FabH by the newly developed compound SB418011 was very potent, with an IC(50) value of 0.016 microm. SB418011 also inhibited E. coli and H. influenzae FabH with IC(50) values of 1.2 and 0.59 microm, respectively. The availability of purified and characterized S. pneumoniae FabH will greatly aid in structural studies of this class of essential bacterial enzymes and facilitate the identification of small molecule inhibitors of type II fatty acid synthase with the potential to be novel and potent antibacterial agents active against pathogenic bacteria.