The amino acid sequences of the protonmotive cytochrome b from seven representative and phylogenetically diverse species have been compared to identify protein regions or segments that are conserved during evolution. The sequences analyzed included both prokaryotic and eukaryotic examples as well as mitochondrial cytochrome b and chloroplast b6 proteins. The principal conclusion from these analyses is that there are five protein regions--each comprising about 20 amino acid residues--that are consistently conserved during evolution. These domains are evident despite the low density of invariant residues. The two most highly conserved regions, spanning approximately consensus residues 130-150 and 270-290, are located in extramembrane loops and are hypothesized to constitute part of the Qo reaction center. The intramembrane, hydrophobic protein regions containing the heme-ligating histidines are also conserved during evolution. It was found, however, that the conservation of the protein segments extramembrane to the histidine residues ligating the low potential b566 heme group showed a higher degree of sequence conservation. The location of these conserved regions suggests that these extramembrane segments are also involved in forming the Qo reaction center. A protein segment putatively constituting a portion of the Qi reaction center, located approximately in the region spanned by consensus residues 20-40, is conserved in species as divergent as mouse and Rhodobacter. This region of the protein shows substantially less sequence conservation in the chloroplast cytochrome b6. The catalytic role of these conserved regions is strongly supported by locations of residues that are altered in mutants resistant to inhibitors of cytochrome b electron transport.