Myostatin, a member of the TGF-β superfamily, is a negative regulator of muscle growth. This study investigates the mechanism by which myostatin controls muscle precursor cell proliferation. The results show that myostatin inhibits the proliferation of C2C12 myoblasts in a dose-dependent manner, with half-maximal inhibition occurring at approximately 2.5 μg/ml. FACS analysis reveals that myostatin prevents the progression of myoblasts from the G1 to S-phase of the cell cycle. Western blot analysis indicates that myostatin up-regulates p21 (Waf1, Cip1), a cyclin-dependent kinase inhibitor (CKI), and decreases the levels and activity of Cdk2 protein in myoblasts. Additionally, myostatin treatment results in the predominance of the hypophosphorylated form of Rb protein. These findings suggest that myostatin signaling leads to increased p21 expression and decreased Cdk2 protein and activity, resulting in the accumulation of hypophosphorylated Rb protein and the arrest of myoblasts in the G1-phase of the cell cycle. The authors propose that the generalized muscular hyperplasia observed in animals lacking functional myostatin is due to deregulated myoblast proliferation.Myostatin, a member of the TGF-β superfamily, is a negative regulator of muscle growth. This study investigates the mechanism by which myostatin controls muscle precursor cell proliferation. The results show that myostatin inhibits the proliferation of C2C12 myoblasts in a dose-dependent manner, with half-maximal inhibition occurring at approximately 2.5 μg/ml. FACS analysis reveals that myostatin prevents the progression of myoblasts from the G1 to S-phase of the cell cycle. Western blot analysis indicates that myostatin up-regulates p21 (Waf1, Cip1), a cyclin-dependent kinase inhibitor (CKI), and decreases the levels and activity of Cdk2 protein in myoblasts. Additionally, myostatin treatment results in the predominance of the hypophosphorylated form of Rb protein. These findings suggest that myostatin signaling leads to increased p21 expression and decreased Cdk2 protein and activity, resulting in the accumulation of hypophosphorylated Rb protein and the arrest of myoblasts in the G1-phase of the cell cycle. The authors propose that the generalized muscular hyperplasia observed in animals lacking functional myostatin is due to deregulated myoblast proliferation.