Although in mitochondria, Escherichia coli and Rhodobacter capsulatus the H+-transhydrogenases are intrinsic membrane proteins, in Rhodospirillum rubrum a water-soluble component (Th(s)) and a membrane-bound component are together required for activity. Th, was selectively removed from chromatophore membranes of Rhs. rubrum and was purified to homogeneity by precipitation with (NH4)2SO4 and ion-exchange, affinity dye and gel exclusion chromatography. The latter indicated an M(r) of approx. 74 000 under non-denaturing conditions but analysis of the pure protein by SDS-PAGE revealed a single polypeptide, M(r) 43 000. Antibodies against this polypeptide inhibited transhydrogenase activity of chromatophores and decreased the capacity of Th(s) to restore activity to depleted membranes. They reacted with a polypeptide of M(r) 43000 in crude cell extract, chromatophore membranes and chromatophore washings but not with transhydrogenase polypeptides from the membranes of E. coli, Rb. capsulatus or animal mitochondria. The N-terminal amino acid sequence of the 43 000 polypeptide was strongly homologous with the reported N-terminal regions of mitochondrial transhydrogenase and the alpha-subunit of the E. coli protein. The break between the alpha and beta-polypeptides of E. coli transhydrogenase is such that both components are membrane-associated. In contrast, these results suggest that in the Rhs. rubrum enzyme Th(s) has been formed by a break closer to the N-terminus, thus avoiding the putative trans-membrane helical segments and yielding a relatively hydrophilic subunit, which is water-soluble. There is a predicted similarity between Th(s) and the reported sequence of alanine dehydrogenase from Bacillus but Th(s) did not have any alanine dehydrogenase activity.