Chickens fed on semi-purified low (7%) or high (61%) protein-energy diets for 14 or 17 d were used for determinations of oxidative phosphorylation and specific amounts of mitochondrial protein in liver. The ADP: oxygen (ADP:O) values obtained when pyruvate + malate were used as substrates were significantly reduced in the high-protein-fed group after the 4th day compared with those for the group fed the low-protein diet, while the differences in ADP:O values between the two treatments when L-glutamate was used as substrate were found to be significant on the 14th day. At any feeding period no significant differences in ADP:O values were observed between the two groups when α-ketoglutarate, malate, or octanoate + malate were used as substrates, nor in specific amounts of mitochondrial protein in liver. The dependency of the pyruvate + malate-supported respiration rate on the temperature in the reaction medium was also determined. The results of an Arrhenius plot showed that transition temperatures, and the lower and upper energies of activation, were similar for the groups fed on low- and high-protein diets. Furthermore, no morphological changes in mitochondria were observed among chickens fed on diets with various protein levels for 14 d. From these results we concluded that the reduction of ADP: O value with pyruvate + malate or L-glutamate substrates in chickens fed on a high-protein diet was substrate-specific, and was not due to functional damage to the respiratory chain for electron flow from NAD-linked substrates to the ubiquinone pool, nor to modulation of properties of the inner mitochondrial membrane.