The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta, organized as an alpha(2)beta(2) tetramer, The protein contains three recognizable domains, of which domain II is the transmembrane region of the molecule containing the pathway for proton translocation, Domain II is composed of four transmembrane helices at the carboxyl-terminus of the alpha subunit and either eight or nine transmembrane helices at the amino-terminal region of the beta subunit. We have introduced pairs of cysteine residues into a cysteine-free transhydrogenase by site-directed mutagenesis. Disulfide bond formation between some of these cysteine residues occurred spontaneously or on treatment with cupric 1,10-phenanthrolinate. Analysis of crosslinked products confirmed that there are nine transmembrane helices in the domain II region of the beta subunit. The proximity to one another of several of the transmembrane helices was determined. Thus, helices 2 and 4 are close to helix 6 (nomenclature of Meuller and Rydstrom, J. Biol. Chem. 274, 19072-19080, 1999), and helix 3 and the carboxyl-terminal eight residues of the alpha subunit are close to helix 7, In the alpha(2)beta(2) tetramer, helices 2 and 4 of one alpha subunit are close to the same pair of transmembrane helices of the other a subunit, and helix 6 of one beta subunit is close to helix 6 of the other beta subunit,