D(--)-beta-Hydroxybutyrate dehydrogenase of beef heart mitochondria is inhibited by modifiers of disulfides, thiols, and vicinal dithiols [Phelps, D. C., & Hatefi, Y. (1981) Biochemistry 20 (preceding paper in this issue)]. The vicinal dithiol can be reversibly oxidized by diamide, resulting in activity inhibition, and rereduced by dithiothreitol, resulting in reactivation. The diamide-treated enzyme can no longer be irreversibly inhibited by N-ethylmaleimide, indicating the absence of an essential sulfhydryl group other than the vicinal dithiol. beta-Hydroxybutyrate dehydrogenase also appears to contain essential arginyl residues modifiable by phenylglyoxal or butanedione, and essential residue(s) modifiable at pH 6.0 by diethyl pyrocarbonate. Substrates protect against inhibitions by butanedione, phenylglyoxal, and diethyl pyrocarbonate, suggesting that the essential, modifiable residues are at or near the substrate binding sites. On the basis of these results and pH profiles, tentative mechanisms have been proposed for the oxidation of beta-hydroxybutyrate and the reduction of acetoacetate, involving the participation of the essential residues described above.