The purpose of the present study was to examine the potential effect of IFN-γ (interferon-γ) on the cellular content and phosphorylation of PKB (protein kinase B), p70S6k (p70 S6 kinase) and MAPK (mitogen-activated protein kinase), and on the ability of insulin to stimulate the glucose uptake and protein synthesis in mouse C2C12 myotubes. Insulin (100 nmol/l) stimulated glucose uptake in C2C12 myotubes by 203.4%. Glucose uptake in cells differentiated in the presence of IFN-γ (10 ng/ml) was increased by 165.8% and was not further significantly modified by the addition of insulin (183.4% of control value). Insulin increased the rate of protein synthesis by 198.8%. The basal rate of protein synthesis was not affected by IFN-γ; however, this cytokine abolished the insulin effect. Cellular levels of PKB, p70S6k, p42MAPK and p44MAPK were not modified by IFN-γ. Insulin caused the phosphorylation of PKB and the activation of p70S6k, but not p42MAPK and p44MAPK. In cells differentiated in the presence of IFN-γ, the insulin-mediated PKB phosphorylation was significantly diminished, whereas the phosphorylation of p70S6k was completely prevented. Pretreatment of C2C12 myogenic cells with IFN-γ led to the marked increase in p42MAPK phosphorylation. Exposure of C2C12 myoblasts to IFN-γ impaired MyoD and myogenin expression and decreased the fusion index on the fifth day of differentiation. In conclusion, (i) IFN-γ present in the extracellular environment during C2C12 myoblast differentiation prevents the stimulatory action of insulin on protein synthesis; (ii) IFN-γ-induced insulin resistance of protein synthesis in myogenic cells can be associated with the decreased phosphorylation of PKB and p70S6k, as well as with the augmented basal phosphorylation of p42MAPK; (iii) this cytokine effect can be partly explained by alterations in the differentiation process.