Abstract The reaction mechanism of human placental estradiol dehydrogenase (17β-estradiol:NAD-oxidoreductase, EC 1.1.1.62) has been investigated by measuring the rate of isotopic exchange between substrate-product pairs while varying concentrations of unlabeled reactants. The results show that the reaction is random with respect to order of substrate addition and that binary complex formation involving either cosubstrate or steroid may occur. Substitution of NADP:NADPH for NAD:NADH produces a marked increase in steroid exchange rates suggesting a decrease in steroid Michaelis constants. Inequality of cosubstrate and steroid exchange rates indicates that chemical transformation is not a rate-limiting step in the over-all reaction. No abortive complex formation was detected.