The effect of dietary protein and fat levels on cardiac mitochondrial oxidative phosphorylation was assessed polarographically. Weanling rats were fed on semi-purified diets containing different protein levels (10, 30, 50 and 70%) on a gross energy basis (PGE) for 9, 23 and 58 d. Cardiac mitochondria isolated from rats fed on a 70% PGE diet for 23 d exhibited significantly reduced ADP:oxygen (ADP:O) values compared with mitochondria from rats fed on a low-protein diet. Feeding low-protein diets for 58 d increased the ADP: O value. When the dietary fat level was altered to provide (% PGE: % fat-energy): 30:14, 30:30, 70:14, 70:30, feeding 70% PGE diets reduced the ADP:O value compared with the 30% PGE level, but no difference was observed between low-fat and high-fat groups. These results indicate that the impaired ADP: O value for rats fed on very-high-protein diets was not due to the dietary fat level but that the level of dietary protein is an important determinant of oxidative phosphorylation in rat heart mitochondria.