The mitochondrial energy-transducing nicotinamide nucleotide transhydrogenase is a homodimer of monomer M(r) = 109,065. It is composed of a 430-residue-long N-terminal hydrophilic domain, a 400-residue-long central hydrophobic domain, and a 200-residue-long C-terminal hydrophilic domain. The N- and the C-terminal hydrophilic domains extend into the mitochondrial matrix, and the central hydrophobic domain intercalates into the inner membrane. Equilibrium binding of [14C]NADH and [14C]NADPH was carried out to the purified transhydrogenase, transhydrogenase modified with N,N'-dicyclohexylcarbodiimide (DCCD) at the NADH-protectable Glu257, and to proteolytic fragments corresponding to dimers of the N-terminal and the C-terminal hydrophilic domains. The intact enzyme bound 1 mol of [14C]NADH or [14C]NADPH per dimer with Kd values of 9.5 and 5.7 microM, respectively. The 90% DCCD-inhibited enzyme bound [14C]NADPH with undiminished capacity and [14C]NADH with 10% capacity as compared with unmodified enzyme. The Kd values were unaltered. The N-terminal fragment bound [14C]NADH at 0.76 mol/dimer with unaltered Kd but did not bind [14C]NADPH. The C-terminal fragment bound 0.32 mol of [14C]NADPH/dimer with unaltered Kd but did not bind [14C]NADH. These results are consistent with our previous assignment of the NAD(H) and the NADP(H) binding sites to the N- and the C-terminal hydrophilic domains, respectively. The binding stoichiometries of 1 mol each of [14C]NADH and [14C]NADPH/dimeric transhydrogenase suggest half-of-the-sites reactivity, which is consistent with our previous findings that 100% inhibition by either p-[3H]fluorosulfonylbenzoyl-5'-adenosine or [14C]DCCD involved the incorporation of 1 mol of inhibitor/dimeric enzyme. We had shown earlier that N-(ethoxycarbonyl)2-ethoxy-1,2-dihydroquinoline (EEDQ) inhibits the transhydrogenase, apparently at the NAD binding site, but differently from DCCD. The enzyme was protected by 5'-AMP strongly against DCCD and weakly against EEDQ, whereas it was protected by NMNH strongly against EEDQ and not at all against DCCD. We have now determined the EEDQ binding sites to be Glu232 and Glu880. The different mononucleotide protections against DCCD and EEDQ suggest that the AMP moiety of NAD binds near Glu257 and its NMN portion near Glu232. They also suggest that the NMN moiety of NADP may bind near Glu880.