The human malaria parasite Plasmodium falciparum synthesizes fatty acids by using a type II synthase that is structurally different from the type I system found in eukaryotes. Because of this difference and the vital role of fatty acids, the enzymes involved in fatty acid biosynthesis of P. falciparum represent interesting targets for the development of new antimalarial drugs. ␤-Ketoacyl-acyl carrier protein (ACP) synthase (PfFabBF), being the only elongating ␤-ketoacyl-ACP synthase in P. falciparum, is a potential candidate for inhibition. In this study we present the cloning, expression, purification, and characterization of PfFabBF. Soluble protein was obtained when PfFabBF was expressed as a NusA fusion protein in Escherichia coli BL21(DE3)-CodonPlus-RIL cells under conditions of osmotic stress. The fusion protein was purified by affinity and ion exchange chromatography. Various acyl-P. falciparum acyl carrier protein (PfACP) substrates were tested for their specific activities, and their kinetic parameters were determined. Activity of PfFabBF was highest with C 4:0 -to C 10:0 -acyl-PfACPs and decreased with use of longer chain acyl-PfACPs. Consistent with the fatty acid synthesis profile found in the parasite cell, no activity could be detected with C 16:0 -PfACP, indicating that the enzyme is lacking the capability of elongating acyl chains that are longer than 14 carbon atoms. PfFabBF was found to be specific for acyl-PfACPs, and it displayed much lower activities with the corresponding acyl-CoAs. Furthermore, PfFabBF was shown to be sensitive to cerulenin and thiolactomycin, known inhibitors of ␤-ketoacyl-ACP synthases. These results represent an important step toward the evaluation of P. falciparum ␤-ketoacyl-ACP synthase as a novel antimalaria target.Malaria is one of the world's most important infectious diseases in terms of both mortality and morbidity. The consensus is that 0.5 billion clinical attacks take place every year, including 2-3 million severe attacks (1-3). It is assumed that the disease claims more than 1 million lives annually and that most of these deaths occur in African children, but the true number might be much higher. Chloroquine, which used to be the first line treatment for malaria, now fails everywhere. Emerging resistance to drugs introduced to replace chloroquine, such as sulfadoxine-pyrimethamine, reinforces the need for new, selective, and affordable drugs against the parasite (1). Of the four causative agents of malaria, i.e. Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae, P. falciparum is the most dangerous. The recent completion of the genome sequencing of P. falciparum allowed the identification of highly promising pathways in the parasite (4). One of the most interesting discoveries was the presence of a complete type II fatty acid biosynthesis pathway (FAS 2 -II) (5, 6). FAS-II is found in bacteria and plants and is structurally very different from the FAS-I system found in most eukaryotes. In FAS-I, the biosynthetic enzymes are int...