Experimental analysis of the protonmotive activity of reconstituted cytochrome c oxidase from beef heart reveals the following features: (1) The observed H+:e- ratio for redox-linked proton ejection from oxidase vesicles is variable, being affected by various effectors that also influence the catalytic process. (2) Proton ejection appears to be associated with electron transfer from heme a (+CuA) to heme a3 (+CuB). (3) Chemical modification studies contribute to indentification of proton-conduction pathways in the protein and/or residues involved in the coupling process between redox and protonmotive activity. In intact rat liver mitochondria, under physiological conditions of dehydrogenase activity and delta microH+ generation by the respiratory chain cytochrome oxidase, does not appear to contribute significant H+ pumping. The relevance of what is observed is discussed in terms of possible mechanisms and physiological role.