The purposes of this study were to compare the deoxygenation patterns of the vastus lateralis (VL) and the lateral head of gastrocnemius (GL) and examine the relationship between the muscle oxygenation level and pulmonary oxygen uptake (VO(2)) during graded treadmill exercise. Changes in oxygenation in each muscle were measured using near infrared spectroscopy (NIRS). Eight healthy male subjects participated in this study. Two NIRS probes were placed on VL and GL, and thereafter the leg arteries were occluded in all subjects to enable normalization of the NIR signals. The subjects then walked at 4 km x h(-1) and 6 km x h(-1), and then ran continuously at speeds ranging from 8 km x h(-1) to 16 km x h(-1). The muscle oxygenation level was defined as being 100% at rest and 0% at its lowest value during occlusion. Pulmonary VO(2) was measured using indirect calorimetry. After the subjects had started walking, the muscle oxygenation in VL increased and exceeded the level at rest. Thereafter, the muscle oxygenation in both muscles decreased in relation to the increase in speed (P < 0.001). A significant difference in the level of muscle oxygenation between VL and GL was found at speeds of 10 km x h(-1) and 12 km x h(-1) (P < 0.05). The muscle oxygenation level at 16 km x h(-1) was [mean (SEM)] 51.9 (4.6)% in VL and 52.8 (3.6)% in GL. There was a negative relationship between pulmonary VO(2) and the muscle oxygenation level (VL: r=-0.803 to -0.986; GL: r=-0.848 to -0.963, P < 0.05). We concluded that the pattern of deoxygenation between VL and GL was somewhat different and that the muscle oxygenation level was associated with pulmonary VO(2).