Endogenous fatty acids are synthesized in all organisms in a pathway catalyzed by the fatty acid synthase complex. In bacteria, fatty acid synthase is made up of several independent cytoplasmic enzymes, each catalyzing one specific reaction. The number of the structurally independent proteins of the synthase complex is species-dependent. The initiation of each elongation step in the fatty acid synthesis cycle requires the transfer of a malonyl moiety from the respective CoA thioester to the –SH group of the phosphopantetheine arm of the acyl carrier protein. Vibrio cholerae, a noninvasive gram-negative bacterium and the causative agent of the diarrheal disease cholera, is serologically classified as belonging to the O antigenic group. We have determined 1.7 Angstrom resolution crystal structure of an acyl carrier protein S-malonyltransferase from the V. cholerae Ol biovar eltor str. N16961. The overall alpha/beta type fold of the protein can be described in terms of a large subunit (residues 1-129 and 206-312) and a small subunit (residues 130-205). The large subunit contains a short four-stranded parallel beta-sheet and twelve alpha helices, whereas the small subunit is made up of a longer four-stranded antiparallel beta-sheet and two alpha helices. The active site is located between two subunits. The hydroxyl of the nucleophilic Ser-96 is located within the large subunit and is within a hydrogen bonding distance of His-206 and Arg-122. The Gln-255 and not a carboxyl acid as in the serine hydrolases completes the catalytic triad. Thus, it can be assumed that in the acyl carrier protein S-malonyltransferase from V. cholerae the acylation step and subsequent transfer of the acyl moiety proceed via tetrahedral intermediate as in serine-dependent proteinases. The molecule of N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) was modeled in the vicinity of the Ser-96 of each of two molecules in the asymmetric unit. In co-crystallization experiments with ligands, it has been shown that CAPS is unfavorable buffer. Optimization of the crystallization conditions for a binary complex between acyl carrier protein S-malonyltransferase and its substrate or inhibitor is underway. Determination of the crystal structure of the binary complex will shed light on mechanism of reaction and propose drug design strategy.