Anaerobic capacity as measured by the maximal or peak oxygen deficit is greater during uphill than during horizontal running. The objective of this study was to determine whether the greater peak oxygen deficit determined during uphill compared with horizontal running is related to greater muscle volume or mass activated in the lower extremity. The peak oxygen deficit in 12 subjects was determined during supramaximal treadmill running at 0 and 10% grade. Exercise-induced contrast shifts in magnetic resonance images were obtained before and after exercise and used to determine the percentage of muscle volume activated. The mean peak oxygen deficit determined for uphill running [2.96 +/- 0.63 (SD) liters or 49 +/- 6 ml/kg] was significantly greater (P < 0.05) than for horizontal running (2.45 +/- 0.51 liters or 41 +/- 7 ml/kg) by 21%. The mean percentage of muscle volume activated for uphill running [73.1 +/- 7. 4% (SD)] was significantly greater (P < 0.05) than for horizontal running (67.0 +/- 8.3%) by 9%. The differences in peak oxygen deficit (liters) between uphill and horizontal running were significantly related (y = 8.05 x 10(-4)x + 0.35; r = 0.63, SE of estimate = 0.29 liter, P < 0.05) to the differences in the active muscle volume (cm3) in the lower extremity. We conclude that the higher peak oxygen deficit during uphill compared with horizontal running is due in part to increased mass of skeletal muscle activated in the lower extremity.