Permanent withdrawal of skeletal myoblasts from the cell cycle precedes differentiation. We examined the changes of protein levels of cell cycle regulators and the activities of CDKs in differentiating (by serum deprivation) or in differentiation-inhibited (by serum deprivation + TGF-beta 1 treated) C2C12 skeletal myocytes, Regardless of differentiation, protein levels of most cyclins declined over time while this effect was delayed slightly by TGF-beta 1 for cyclins D1 and F, Although the protein levels of CDKs gradually decreased over time, the activities of CDK2 and cdc2 decreased dramatically between 0 and 12 h after serum deprivation in both groups, Decreased CDK2 and cdc2 activities were temporally related to decreased DNA synthesis. While the p27(KIP1) protein increased in both groups, the p21(CIP1) protein increased only in differentiating cells. III vivo, protein expressions of cyclins, CDKs, and p21(CIP1) were high in fetal, but almost undetectable in adult skeletal muscle. In contrast, the levels of p27(KIP1) protein in skeletal muscle were high throughout development. Thus, changes of cell cycle regulators in differentiating C2C12 myocytes paralleled those during skeletal muscle development of mice, These results suggest that the rapid reduction of DNA synthesis and activities of CDKs slow reduction of protein levels of cyclins and CDKs, and that the increase of p27(KIP1) are programmed mechanisms upon mitogen deprivation regardless of differentiation in skeletal myocytes.