OBJECTIVES: Growing evidence supports hip muscle activation and strengthening exercise prescription to prevent and treat various lower limb injuries. Common prescriptions include single-legged and double-legged squatting, with and without a Swiss ball. We aimed to establish the effect of varying forms of squatting exercises on gluteal muscle activation. DESIGN AND SETTING: Observational laboratory study. PARTICIPANTS: Nineteen (11 male) healthy participants (28.4 +/- 2.7 years old) were compared using one-way repeated measures analysis of variance. MAIN OUTCOME MEASURES: Surface electromyography (EMG) measures of gluteus medius (GMed) and gluteus maximus (GMax) during the isometric phase of single-legged and double-legged squatting, with and without a Swiss ball. RESULTS: A greater percentage of maximal voluntary contraction (%MVC) during single-legged squatting was found compared to double-legged squatting for GMed (42 versus 9%MVC, p < 0.001) and GMax (35 versus 14%MVC, p < 0.001). Additionally, the Swiss ball increased GMax activity (42 versus 35%MVC, p = 0.026) and demonstrated a trend toward increased GMed activity (46 versus 42%MVC, p = 0.075) during the single-legged squat. CONCLUSIONS: These results indicate single-legged squatting may be more appropriate than double-legged squatting to facilitate strength gains of GMed and GMax. Additionally, the Swiss ball may be a useful adjunct to target gluteal muscle strengthening during single-legged squatting.