Skeletal muscle mitochondria are implicated with age-related loss of function and insulin resistance. We examined the effects of exercise on skeletal muscle mitochondria in older (age = 67.3 ± 0.6 years) men (n = 5) and women (n = 3). Similar increases in (p <.01) cardiolipin (88.2 ± 9.0 to 130.6 ± 7.5 μg/mU creatine kinase activity [CK]) and the total mitochondrial DNA (1264 ± 170 to 1895 ± 273 copies per diploid of nuclear genome) reflected increased mitochondria content. Succinate oxidase activity, complexes 2–4 of the electron transport chain (ETC), increased from 0.13 ± 0.02 to 0.20 ± 0.02 U/mU CK (p <.01). This improvement was more pronounced (p <.05) in subsarcolemmal (127 ± 48%) compared to intermyofibrillar (56 ± 12%) mitochondria. NADH oxidase activity, representing total ETC activity, increased from 0.51 ± 0.09 to 1.00 ± 0.09 U/mU CK (p <.01). In conclusion, exercise enhances mitochondria ETC activity in older human skeletal muscle, particularly in subsarcolemmal mitochondria, which is likely related to the concomitant increases in mitochondrial biogenesis.