The purpose of this study was to compare muscle activity of the lower limb during treadmill running under five different gradient conditions (level, left and right inclines, downward and upward). All inclines were of 14% grade and tilted toward the left, the right, downward and upward directions of the runner. Twelve young healthy males ran at 2.8 m/s. Electromyographic activities of the following seven muscles-gluteus maximus, biceps femoris, rectus femoris, vastus lateralis, tibialis anterior, medial gastrocnemius, and solues-were measured using bipolar surface electrodes during treadmill running under five different conditions. Results showed that left and right inclined surfaces had only little influence on muscular activity. However, for all of the muscles measured, the activity of the lower-positioned leg was greater than that of the higher-positioned leg. During the upward incline trial, all the muscles showed greater activity than for the other trials. The downward slope tended to give the anterior muscles greater activity, and the posterior muscles less activity, compared to level running.