Electromyographic (EMG) activity in the human masseter muscle was registered from six different sites, in the anterior, middle, and posterior regions of the superficial and deep layers of the muscle, during static clenching tasks (intercuspal and incisal), selected jaw movements (alternating protrusion/retrusion, right/left latero-deviation, and open/close excursions), and unilateral chewing on right and left sides. Peak-EMG amplitudes and the timing of the peaks were compared. Activity in the regions of the deep masseter was either higher (in mastication and intercuspal open/close excursions) or lower (incisal clenching) than the activities in the superficial masseter. Superficial and deep masseter also differed in their timing of peak EMG: During chewing, peak activity passed from superficial to deep in the balancing-side muscle, and from deep to superficial on the chewing side. During free latero-deviations, peak activity started in the deep masseter, when the jaw moved to the right side (i.e., the side of the muscle), and then passed to the superficial regions, after the jaw movement was reversed to the left side. In addition, within the deep masseter there existed clear anteroposterior differences in activation level (during incisal clenching and open/close excursions) and in timing (during latero-deviation). Such a differentiation of activity was not found in the superficial masseter.