Rat liver DT-diaphorase is inactivated by 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), following pseudo-first-order kinetics. A double-reciprocal plot of 1/kobs versus 1/[5'-FSBA] yields a straight line with a positive y intercept, indicative of reversible binding of the inhibitor before an irreversible incorporation. The dissociation constant (Kd) for the initial reversible enzyme-inhibitor complex is estimated at 2.86 mM with k2 = 0.22 min-1 (at pH 7.5 and 25 degrees). A stoichiometry of 2 mol of 5'-FSBA bound/mol of enzyme (i.e., 1 mol of the inhibitor bound/mol of subunit), at 100% inactivation, was determined from inactivation kinetics and from incorporation studies using 5'-[p-(fluorosulfonyl)benzoyl]-[14C]-adenosine. The irreversible inactivation as well as the covalent incorporation could be completely prevented by the presence of NAD(P)H during the incubation. These results indicate that 5'-FSBA inactivated DT-diaphorase by occupying its NAD(P)H binding site. Reverse phase high pressure liquid chromatography of tryptic digests of [14C]5'-FSBA-labeled DT-diaphorase revealed one radioactive peak containing two comigrating peptides. They are 146I-T-T-G-G-S-G-S-M-Y155 and 262S-I-P-A-D-N-Q-I-K270. By comparison of these sequences to those of the nucleotide binding sites of several kinases and dehydrogenases, it is suggested that the peptide I-T-T-G-G-S-G-S-M-Y is the one modified by 5'-FSBA and would be predicted to be the region where the pyrophosphate group of NAD(P)H binds.