Cytochrome cbb(3) belongs to the superfamily of respiratory heme-copper oxidases that couple the reduction of molecular oxygen to proton translocation across the bacterial or mitochondrial membrane. The cbb(3)-type enzymes are found only in bacteria, and are both structurally and functionally the most distant from their mitochondrial counterparts. The mechanistic H(+)/e(-) stoichiometry of proton translocation in these cbb(3)-type cytochrome c oxidases has remained controversial. A stoichiometric efficiency of only one-half that of the mitochondrial aa(3)-type enzyme was recently proposed to be related to adaptation of the organism to microaerobic environments. Here, proton translocation by the Rhodobacter sphaeroides enzyme was studied using purified cytochrome cbb(3) reconstituted into liposomes. An H(+)/e(-) stoichiometry of proton translocation close to unity was observed using the oxygen pulse method, but solely in conditions in which the vast majority of the enzyme was fully reduced in the anaerobic state before the O(2) pulse. These data were compared with results using whole cells or spheroplasts, and the discrepancies in the literature data were discussed. Our results suggest that a proton-pumping efficiency of 1 H(+)/e(-) may be achieved using the single-proton uptake pathway identified in the structure of cytochrome cbb(3). The mechanism of proton pumping thus differs from that of the aa(3)-type oxidases of mitochondria and bacteria.