Imbalances in the firing pattern and contraction intensity of the vastus medialis oblique (VMO) and the vastus lateralis (VL) have been considered important factors contributing to patellofemoral joint dysfunction. Vastus medialis oblique and vastus lateralis electromyographic (EMG) activity were measured for 15 individuals without patellofemoral pain (asymptomatic group) and 13 subjects with patellofemoral pain (symptomatic group) while ascending and descending steps. The peak VMO/VL ratios of EMG activity and the difference in peak VMO and VL onset times were measured. Two-way mixed-model analyses of variance (ANOVA) were used to determine the main effects of group (asymptomatic and symptomatic), phase (concentric phase ascending and two eccentric phases descending stairs), and the interaction between group and phase. The ANOVAs indicated no difference between groups for the peak VMO/VL EMG ratio or for the onset timing between peak VMO and VL muscle activity. Combining groups, the peak VMO/VL EMG ratio was less for the eccentric weight acceptance phase of descent compared with the concentric phase of ascent. These findings suggest no differences between asymptomatic and symptomatic individuals, but differences may exist between concentric and eccentric VMO/VL ratios. Further research is needed to determine if VMO and VL muscle imbalances contribute to patellofemoral dysfunction.