Abstract The enzyme, β-ketoacyl acyl carrier protein (ACP) synthetase, which catalyzes the formation of acetoacetyl-ACP from acetyl-ACP and malonyl-ACP, has been purified to homogeneity from Escherichia coli. The molecular weight, determined from equilibrium centrifugation studies, is 66,000. Amino acid analysis after performic acid oxidation indicates that it contains 8 moles of cysteic acid per mole of protein. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) after protein denaturation with sodium dodecyl sulfate indicates 7.2 —SH groups per mole of protein. The enzyme is inhibited by iodoacetamide when 1 mole of cysteine per mole of protein is alkylated, and acetyl-ACP protects the enzyme against alkylation. The enzyme reacts with acetylACP to form an acetyl enzyme intermediate, and the properties of this intermediate suggest that the acetyl group is bound to a cysteine residue as an acyl thioester. Acetyl enzyme is active in transferring the acetyl group either to ACP to form acetyl-ACP or to malonyl-ACP to form acetoacetyl-ACP.