Abstract Although highly purified preparations of β-ketoacyl acyl carrier protein (ACP) reductase are obtained from extracts of Escherichia coli, purification is hampered by the enzyme's instability in dilute solution. An investigation of this phenomenon revealed that concentrations of TPNH and TPN+ below 1 mm and those of multivalent anions between 0.2 m and 1 m could protect the active form of the enzyme by binding to the protein and thereby stabilizing its active form. Enzyme inactivation occurred in two stages, the first being reversible by TPNH, TPN+, and multivalent anions, and the second being irreversible by these agents. In these studies potassium phosphate acted as an inhibitor of TPNH oxidation, while potassium chloride failed to alter the reaction rate, indicating that TPNH, TPN+, and phosphate may be bound to the same site (possibly the catalytic site) of the enzyme. From a comparative kinetic study with a number of model substrates, it was concluded that the substrate, acetoacetyl-ACP was bound to β-ketoacyl-ACP reductase through its protein moiety, the pantoyl residue of the 4'-phosphopantetheine prosthetic group, and the β-ketoester group. ACP was a competitive inhibitor of the reduction of acetoacetyl-ACP, acetoacetyl pantetheine and acetoacetyl-N-acetyl-cysteamine, emphasizing the importance of the ACP-protein-enzyme interaction for substrate binding and demonstrating the inaccessibility of the catalytic site for model substrates once the ACP-protein is bound to the enzyme. Since the enzyme reduced ethyl thiolacetoacetate and ethyl acetoacetate, we concluded that the cysteaminyl residue and the thioester linkage of the acetoacetyl substrate were not absolutely necessary for the substrate-enzyme binding. However, the absence of the cysteaminyl residue in the substrate significantly decreased the Vmax values. The fact that acetoacetyl-ACP in its enolate form would apparently not bind to the enzyme suggests that the keto form of the substrate is directly reduced in this reaction.