The pyridine nucleotide transhydrogenase (EC 1.6.1.1) carries out transmembrane proton translocation coupled to transfer of a hydride equivalent between NAD+ and NADP+. Mutations were made in histidine-91 of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli. This amino acid is the only conserved charged residue in the transmembrane domains of this enzyme and thus potentially is involved in proton translocation by the transhydrogenase. The mutant beta H91N retained 80% of the hydride transfer activity while proton translocation was reduced to 7%. This behavior is consistent with a role for beta His91 in the proton translocation pathway. Other mutations at this residue affected the conformation of the enzyme. Thus, the enzyme in mutants beta H91C, beta H91T, and beta H91S was unable to undergo the conformational change that occurred on binding of the substrates NADP+ or NADPH. By contrast, the enzyme in the beta H91K mutant was present in the NADP(H)-induced conformation even in the absence of these substrates. Further evidence for the linkage between beta His91 and the conformation of the beta subunit was obtained by labeling the transmembrane domain of the beta subunit with [14C]N,N'-dicyclohexylcarbodiimide (DCCD). Labeling occurred most readily with the enzyme of beta H91K. It is concluded that beta His91 is a component of the proton translocation pathway of the transhydrogenase and that its state of protonation is probably linked to conformational changes induced by binding/debinding of substrates during the catalytic cycle of the enzyme.