Background Age related declines in walking performance may be partly attributable to skeletal muscle mitochondrial dysfunction as mitochondria produce over 90% of ATP needed for movement and the capacity for oxidative phosphorylation decreases with age. Methods Participants were from two studies: an ancillary to the Lifestyle Interventions and Independence for Elders (LIFE) Study (n =33), which recruited lower functioning participants (Short Physical Performance Battery [SPPB], 7.8±1.2), and the Study of Energy and Aging-Pilot (SEA, n =29), which enrolled higher functioning (SPPB, 10.8±1.4). Physical activity was measured objectively using the Actigraph accelerometer (LIFE) and SenseWear Pro armband (SEA). Phosphocreatine recovery following muscle contraction of the quadriceps was measured using 31P magnetic resonance spectroscopy and ATPmax (mM ATP/s) was calculated. Walking performance was defined as time (s) to walk 400m at a usual-pace. The cross-sectional association between mitochondrial function and walking performance was assessed using multivariable linear regression. Results Participants were 77.6±5.3years, 64.2% female and 67.2% white. ATPmax was similar in LIFE vs. SEA (0.52±0.14 vs. 0.55±0.14, p =0.31), despite different function and activity levels (1.6±2.2 vs.77.4±73.3min of moderate activity/day, p <0.01). Higher ATPmax was related to faster walk-time in SEA (r2 =0.19, p =0.02,); but not the LIFE (r2 <0.01, p =0.74) cohort. Conclusions Mitochondrial function was associated with walking performance in higher functioning, active older adults, but not lower functioning, sedentary older adults.