Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by proteases within the N terminus, exposing a new tethered ligand that binds and activates the receptor. Activators of PAR-2 include trypsin and mast cell tryptase. Skeletal myoblasts are known to express PAR-1, a thrombin receptor. The current study was undertaken to determine whether myoblasts express PAR-2. Primary neonatal rat and mouse skeletal myoblast cultures were shown to express PAR-2 in polymerase chain reaction and immunocytochemical studies. Expression of PAR-2 was also demonstrated by immunohistochemistry in developing mouse skeletal muscle in vivo. Trypsin or a synthetic peptide corresponding to the rat PAR-2 tethered ligand caused a dose-dependent elevation in intracellular calcium in cultured rat myoblasts, with an EC(50) of 13 nM or 56 microM, respectively. Studies aimed at identifying the function of PAR-2 in myoblasts demonstrated no effect of the receptor-activating peptide on survival or fusion in serum-deprived myoblasts. The PAR-2-activating peptide did, however, stimulate proliferation of serum-deprived myoblasts. These results demonstrate that skeletal muscle cells express PAR-2, activation of which leads to stimulation of myoblast proliferation.