1. The regulatory mechanism for the hypertrophy of skeletal muscles induced by clenbuterol is unclear. The purpose of the present study was to determine the extent to which transforming growth factor betas (TGFbetas), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), and platelet-derived growth factors (PDGFs) are involved in the hypertrophy of rat masseter muscle induced by clenbuterol. 2. We measured the mRNA expression levels for TGFbetas, FGFs, HGF, and PDGFs in rat masseter muscle hypertrophied by oral administration of clenbuterol for 3 weeks and determined correlations between the weight of masseter muscle and mRNA expression levels by regression analysis. We determined immunolocalizations of TGFbetas and their receptors (TGFbetaRs). 3. The mRNA expression levels for TGFbeta1, 2, and 3, and for PDGF-B demonstrated clenbuterol-induced elevations and positive correlations with the weight of masseter muscle. In particular, TGFbeta1, 2, and 3 showed strong positive correlations (correlation coefficients >0.6). The mRNA expression levels for PDGF-A, FGF-1 and 2, and HGF showed no significant differences between the control and clenbuterol groups, and no significant correlations. TGFbeta1, 2, and 3 were principally localized in the connective tissues interspaced among myofibers, and TGFbetaRI and II were localized in the periphery and sarcoplasm of the myofibers. 4. These results suggest that paracrine actions of TGFbeta1, 2, and 3 via TGFbetaRI and II could be involved in the hypertrophy of rat masseter muscle induced by clenbuterol. This is the first study to document the involvement of TGFbetas in the hypertrophy of skeletal muscles induced by clenbuterol.