The binding of sulfonamides to the active site of horse liver alcohol dehydrogenase has been studied by their effect on affinity labelling and steady state kinetics. Affinity labelling with iodoacetate and BIP has been used to study binding to free enzyme. The unsubstituted sulfonamide, sulfanilamide (I), shows very weak binding compared to the other sulfonamides tested. Most important for binding is the type of substituent attached to the parent sulfonamide, particularly when as in sulfathiazole this is a heterocycle which binds to the catalytic zinc atom of the enzyme. For sulfathiazole the dissociation constant from the enzyme is pH dependent showing two pKa values. The lower at pH 7 is the pKa of the drug itself, while that at pH 9 agrees with the ionization of water bound to the catalytic zinc ion. Steady state kinetics have been carried out at pH 7.0 and 10.0 to examine sulfonamide binding to the enzyme when coenzyme is attached. Both NAD+ and NADH induce substrate competitive sulfonamide binding. Likewise sulfathiazole accelerates the dissociation of NADH from the enzyme and SO Vmax for alcohol oxidation. The latter like stimulation of the affinity labelling reaction with iodoacetate is considered to result from binding of the thiazole ring to the catalytic zinc ion. With all the sulfonamides examined hydrophobic binding and charge are important in determining affinity to the active site and the mode of binding. Sulfonamides containing pyrazole or imidazole rings can be important in alcohol therapy.