The pyridine nucleotide transhydrogenase carries out transmembrane proton translocation coupled to transfer of a hydride ion equivalent between NAD⁺and NADP⁺. Previous workers (E. Holmberget al. Biochemistry33, 7691–7700, 1994; N. A. Glavaset al. Biochemistry34, 7694–7702, 1995) had examined the role in proton translocation of conserved charged residues in the transmembrane domain. This study was extended to examine the role of conserved polar residues of the transmembrane domain. Site-directed mutagenesis of these residues did not produce major effects on hydride transfer or proton translocation activities except in the case of βAsn222. Most mutants of this residue were drastically impaired in these activities. Three phenotypes were recognized. In βN222C both activities were impaired maximally by 70%. The retention of proton translocation indicated that βAsn222 was not directly involved in proton translocation. In βN222H both activities were drastically reduced. Binding of NADP⁺but not of NADPH was impaired. In βN222R, by contrast, NADP⁺remained tightly bound to the mutant transhydrogenase. It is concluded that βAsn222, located in a transmembrane α-helix, is part of the conformational pathway by which NADP(H) binding, which occurs outside of the transmembrane domain, is coupled to proton translocation. Some nonconserved or semiconserved polar residues of the transmembrane domain were also examined by site-directed mutagenesis. Interaction of βGlu124 with the proton translocation pathway is proposed.