Chronic administration of clenbuterol (CB), a lipophilic b2-adrenoceptor (b2-AR) agonist, induces skeletal muscle hypertrophy and slow-to-fast fiber–type transitions in mammalian species, but the mechanism and pathophysiological roles of these changes have not been explored. Here, we examined the effects of CB not only on masseter muscle mass, fiber diameter, and myosin heavy chain (MHC) composition, but also on daily muscle activity, a factor influencing muscle phenotype, by means of electromyogram analysis in rats. MHC transition towards faster isoforms was induced by 2-week CB treatment. In addition, daily duty time was increased at 1 day, 1 week, and 2 weeks after the start of CB treatment and its increase was greater at high activity level (6-fold) than at low activity level (2-fold). In order to examine whether these effects of CB were mediated through muscle or CNS b2-AR stimulation, we compared these effects of CB with those of salbutamol (SB), a hydrophilic b2-AR agonist. SB treatment induced masseter hypertrophy and MHC transition, like CB, but did not increase daily activity. These results suggest that CB-mediated slow-to-fast MHC transition with hypertrophy was induced through direct muscle b2-AR stimulation, but the increase of daily duty time was mediated through the CNS.[Supplementary Figures: available only at http://dx. doi. org/10.1254/jphs. 12271FP]