Mitochondrial enzyme activities and ultrastructure of mitochondria prepared from klotho mutant mice were compared with those in wild-type mice. We also measured the levels of expression of ND1, 51kDa, and 75kDa mRNA associated with the genes encoding NADH dehydrogenase and complex I and that of alpha cardiac myosin heavy chain mRNA in both groups. Mitochondrial NADH oxidoreductase activity was higher in klotho mutant mice during aging than that in wild-type mice. The area of mitochondria per unit area (300µm²) of cell was almost constant from 4 to 7 weeks of age in both groups. A few large mitochondria were scattered between numerous small mitochondria with compact cristae and myofibrils in klotho mice from 5 weeks of age. The levels of ND1 and 75kDa mRNA were slightly high from 7 weeks of age in klotho mutant mice, whereas they were almost constant in wild-type mice, in spite of reduced expression of alpha cardiac myosin heavy chain mRNA. Our results indicate that klotho protein indirectly plays a role in diminished functional adaptability of enzymes in aged heart muscle, and is required for hypertrophy of cardiac mitochondria.