Differentiation of skeletal myoblasts into contractile myotubes is associated with permanent withdrawal from the cell cycle. Little is known about the expression of cell cycle regulating genes during terminal differentiation of muscle cells. We investigated the expression pattern, biological activity, and cellular localization of cyclins and cyclin-dependent kinases during terminal differentiation of the mouse skeletal myogenic cell line C2C12. After induction of differentiation by serum deprivation, cdc2 mRNA levels transiently increased, followed by a down-regulation to undetectable levels within 42 h. In contrast, cdk2 mRNA stayed constant during this period. During differentiation cyclin A, B, and C were down-regulated within 24 h to undetectable levels. Interestingly, cyclin D1/CYL1 mRNA was up-regulated by twofold at 9-12 h after serum deprivation followed by a down-regulation to undetectable levels within 42 h, while cyclin D3/CYL3 mRNA levels remained constant. Restimulation of the differentiated myotube culture with serum reinduced cdc2 as well as cyclin D1/CYL1 mRNA close to the levels observed in dividing myoblasts. At the protein level p34cdc2 was detected in nuclei of proliferating myoblasts and nascent myotubes, but not in mature myotubes. Restimulation with serum-induced p34cdc2 protein in a small minority of unfused myoblasts, but never in myotubes. Histone H1 kinase activity of p34cdc2 decreased during differentiation while p33cdk2 activity did not change. These findings suggest that terminal differentiation of skeletal muscle cells is associated with a differential regulation of cyclins and their associated kinases. Inability to accumulate p34cdc2 protein in response to serum stimulation, despite the induction of its mRNA, in differentiated myotubes may play an important role in maintaining the postmitotic state of skeletal muscle in the presence of high concentrations of growth factors.