Cell differentiation is usually accompanied by irreversible cell cycle exit, which is a critical step for skeletal muscle differentiation. We therefore hypothesise that PD98059 that blocks the MAP kinase kinase (MEK) pathway (proliferation pathway) when administrated to murine C2 skeletal myoblasts will arrest cell cycle and, consequently, enhances differentiation relative to untreated controls. In this study, we aimed to examine this hypothesis using phenotypic differentiation, biochemical assays, flow cytometry and real-time PCR in C2 cells cultured for 48 h in differentiation media only (untreated) or supplemented with either a single dose of 10 ng/ml IGF-I or 20 muM PD98059 for 48 h. Creatine kinase (CK) activity was increased by 7.5-fold (P<0.05) in the presence of PD98059, whereas untreated and IGF-I-treated cells induced 4.5- and 4-fold increase respectively when compared with baseline controls. Increased CK values in the presence of PD98059 were not only associated with myotube formation but also associated with cell cycle arrest in G1 phase (86+/-3.2%; P<0.05). Moreover, the expression of myogenic-specific transcriptional factor mRNAs (MyoD and myogenin) was significantly higher in PD-treated cells (4.7+/-0.15 and 314+/-10.2 ng/reaction respectively; P<0.05) than untreated (2.0+/-0.2 and 233+/-11 ng/reaction respectively) or IGF-treated cells (3.2+/-0.24 and 296+/-16.2 ng/reaction respectively). Unexpectedly, Id3 mRNA, the potent negative regulator of muscle differentiation, was also expressed at significantly higher levels in PD-treated cells (77+/-0.346 ng/reaction; P<0.05) than untreated (49+/-7.7 ng/reaction) or IGF-I-treated cells (47+/-0.7 ng/reaction). Furthermore, expression of the muscle differentiation-specific genes (IGF-binding protein-5, IGF-II receptor and IGF-II) was also increased significantly in PD-treated cells when compared with untreated cells. Phosflow analysis showed a significant increase in the levels of phosphorylation of p38 mitogen-activated protein kinase (49.0+/-6.7%, P<0.05) in PD-treated cells when compared with DM-treated cells (31.7+/-5.7%). These findings uncover a previously unconsidered positive effect of PD98059 on C2 myoblast differentiation and identify the pathway(s) underlying PD-induced C2 myoblast differentiation.