The binding of TNP-ATP (2′ or 3′-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant Kd = 0.2 µM) and one with lower affinity (Kd = 0.9 µM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (Kd = 3.6 µM). The binding of [35S]ATPaS was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (Kd = 7.5 µM) and six at the liver enzyme (Kd = 12 µM). The Paracoccus enzyme had only one binding site (Kd = 16 µM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H+/e- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H+/e- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H+/e- stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature. (Mol Cell Biochem 174: 131–135, 1997)