In this study, we investigated the effect of ultrasound (US) on the activity of commercial cellulase (Celluclast (R) 1.5 L) in the absence and in the presence of a cellulosic substrate (Avicel (R), 2% w.v(-1)) using a central composite rotatable design. Sonication time (10 to 330 s), US intensity (120.6 to 263.7 W cm(-2)), and reaction temperature (25 to 50 degrees C) were varied using a horn-type ultrasound reactor, and endoglucanase (CMCase) and total cellulase (FPase) activities were determined. US intensity had a positive effect on enzyme activity. Under optimal conditions (170 s, 180.8 W cm(-2), and 25 degrees C), CMCase activity was 13% higher than that of the control. In the presence of substrate, CMCase activity increased by 33.87% and K-M reduced by 23% in relation to that of the control. The theoretical yield of cellulose was 42.08%. Cellulase activity can be improved by US treatment to maximize productivity gains and reduce costs in second-generation ethanol production, by the action of a low-frequency ultrasound with a short ultrasonication time of application.