D-3-Hydroxybutyrate dehydrogenase (Pseudomonas lemoignei, EC 1.1.1.30) is subject to substrate inhibition by acetoacetate at concentrations above 5 mM but not by D-3-hydroxybutyrate at concentrations up to 50 mM. NADH causes substrate inhibition at concentrations over 0.1 mM as does NAD. Kinetic analysis suggests that substrate inhibition by acetoacetate is due to its binding to enzyme lacking NADH, a consequence of the ordered bibi mechanism. Substrate inhibition by NADH and NAD arises from binding of these species to a secondary site. This is confirmed by kinetics which indicate that ADP and ATP compete with NAD and NADH at both sites. New analogues of acetoacetate were synthesized to test the specificity requirements of the acetoacetate binding site which has been proposed to contain a hydrogen bond donor and a cation spaced to receive acetoacetate. Both dimethoxyphosphinylacetate and methyl 2-methoxy-phosphinylacetate fulfill the structural requirements and are effective. They thus join methyl acetonylphosphonate as the only known competitive inhibitors for the acetoacetate site, confirming the proposed structure.