The binding of NAD and NADH to electrophoretically pure 3(17)beta-hydroxysteroid dehydrogenase of Pseudomonas testosteroni was determined by Fluorescence spectroscopy and gel filtration. Four moles of cofactor are bound/mol of tetrameric enzyme; the binding sites are equivalent and independent. The dissociation constants for NAD and NADH are 16 and 0.25 micronM, respectively. As measured by gel filtration in the absence of cofactor, 0.4 mol of estradiol-17 beta is bound/mol of tetrameric enzyme. Data obtained from isotope exchange at equilibrium indicate that the binding of the cofactor to the enzyme is favored over the binding of steroid, although each may bind in the absence of the other. The rates of cofactor dissociation from the ternary complexes are slower than the rates of steroid dissociation; cofactor dissociation is probably the rate-limiting step. Cofactor analogs modified in the pyridine moiety are cosubstrates, whereas modified adenine derivatives are not. The enzyme also utilized as substrate a number of potential steroid affinity labels; no enzyme inactivation by these compounds was observed.