The kinetic mechanism for the reduction of acetohexamide catalyzed by carbonyl reductase from rabbit kidney was investigated. The initial velocity and product inhibition studies indicated that the enzymatic reaction follows an ordered Bi Bi mechanism, in which NADPH binds to the enzyme first and NADP leaves last. This kinetic mechanism was confirmed on the basis of the dead-end inhibition by Cibacron Blue and the binding of NADPH and NADP to the free enzyme. However, whether or not coenzyme-induced isomerization is involved in the enzymatic reaction remains to be clarified. In kinetic studies of inhibition of the enzyme by therapeutically active drugs, indomethacin and befunolol were found to be noncompetitive and competitive inhibitors, respectively, with respect to acetohexamide.