The Rhodospirillum rubrum structural gene puh, coding for the photoreaction center H polypeptide, and three other putative genes that surround puh were cloned and sequenced. The deduced 257 amino acid H polypeptide has a molecular weight of 27,909, in close agreement with polyacrylamide gel electrophoresis determination. Hydropathy plots predict a single hydrophobic alpha helix. The H polypeptide of Rhodospirillum rubrum shares only 23% of its residues with all three of the H polypeptides from Rhodopseudomonas viridis, Rhodobacter capsulatus, and Rhodobacter sphaeroides. Despite this apparent low degree of similarity, statistical analysis leaves no doubt about their close relatedness. Interspecies evolutionary distance, assessed by this analysis, confirms the closeness of the two Rhodobacter species, Rhodospirillum rubrum and Rhodopseudomonas viridis being approximately equidistant from them. Three regions of the H polypeptide are highly conserved in all four species. They correspond to known contact points of H with the complex of the other two (L and M) subunits on the cytoplasmic side of the membrane. A glutamic acid residue (H polypeptide residue 177), conserved in the other bacteria and suggested to be involved in the binding of secondary quinone QB, is replaced by serine in Rhodospirillum rubrum. The open reading frames G115, I2372, and I3087 are predicted to, respectively, encode polypeptides of 480, 224, and 155 residues coiled in 10, 2, and 1 transmembrane helices. Open reading frame G115 shares 56% identical residues with F1696, a sequence arranged in the genome of Rhodobacter capsulatus. The gene product of ORF I3087 is predicted to share highly similar sequences with nitrogenase reductase (encoded by nifH) of 11 different bacterial species and is suggested to have a regulatory function.