Based on the amino acid sequence of the N-terminus of the soluble subunit of theRhodospirillum rubrum nicotinamide nucleotide transhydrogenase, two oligonucleotide primers were synthesized and used to amplify the corresponding DNA segment (110 base pairs) by the polymerase chain reaction. Using this PCR product as a probe, one clone with the insert of 6.4kbp was isolated from a genomic library ofR. rubrum and sequenced. This sequence contained three open reading frames, constituting the genesnntA1, nntA2, andnntB of theR. rubrum transhydrogenase operon. The polypeptides encoded by these genes were designated 1, 2, and , respectively, and are considered to be the subunits of theR. rubrum transhydrogenase. The predicted amino acid sequence of the 1 subunit (384 residues; molecular weight 40276) has considerable sequence similarity to the subunit of theEscherichia coli and the N-terminal 43-kDa segment of the bovine transhydrogenases. Like the latter, it has a fold in the corresponding region, and the purified, soluble 1 subunit cross-reacts with antibody to the bovine N-terminal 43-kDa fragment. The predicted amino acid sequence of the subunit of theR. rubrum transhydrogenase (464 residues; molecular weight 47808) has extensive sequence identity with the subunit of theE. coli and the corresponding C-terminal sequence of the bovine transhydrogenases. The chromatophores ofR. rubrum contain a 48-kDa polypeptide, which cross-reacts with antibody to the C-terminal 20-kDa fragment of the bovine transhydrogenase. The predicted amino acid sequence of the 2 subunit of theR. rubrum enzyme (139 residues; molecular weight 14888) has considerable sequence identity in its C-terminal half to the corresponding segments of the bovine and the subunit of theE. coli transhydrogenases.