Step ergometry has become a popular treatment mode in cardiovascular fitness and knee rehabilitation programs. The following study describes the effects of forward stair stepping at 35 and 95 steps/minute and forward and retrograde stair stepping at 60 steps/minute on the mean electromyographic (EMG) activity of the gluteus maximus, rectus femoris, vastus medialis, semimembranosus/semitendinosus, and gastrocnemius muscles. Thirty-three subjects without prior lower extremity pathology participated in the study. Analysis of variance was used to compare mean EMG activity during the knee extension phase of the different stepping conditions. Significant differences (p < 0.05) in mean EMGs of gluteus maximus, rectus femoris, vastus medialis, and gastrocnemius were noted across all cadences. The semimembranosus/semitendinosus EMG activity displayed a single significant difference between the cadences of 35 and 95. The comparison between forward and retrograde stepping identified only one significant difference in mean EMG (semimembranosus/semitendinosus). The effects of cadence on EMG activity should be considered when developing conditioning or rehabilitation programs for the lower extremity. Faster cadences result in increased peak activity of shorter duration for the gluteus maximus, quadriceps, and gastrocnemius. There is no apparent difference in EMG activity of the hamstrings due to cadence. Also, noting the minimal activation that occurs, stair-stepping exercise would not be recommended for strengthening of the hamstrings musculature.