The purpose of this study was to determine whether creatine 5 mM increases protein synthesis in myogenic C₂C₁₂ cells and to identify the signalling cascade(s) implicated. Creatine increased protein synthesis as measured by a larger incorporation of labelled [³⁵S]methionine into sarcoplasmic (P<0.05) and myofibrillar (P<0.01) proteins after 5 days of differentiation. Moreover creatine promoted the fusion of myoblasts since creatine increased the number of nuclei within myotubes by 40% after 96 h of differentiation (P<0.001). To determine how creatine signals, we first analyzed some protein markers affected by creatine. At 96 h of differentiation, creatine enhanced the expression of myosin heavy chain-II (P<0.001), troponin T (P<0.01) and titin (P<0.05) but neither citrate synthase nor lacticodehydrogenase. Because we had previously reported that creatine increased insulin-like growth factor-I (IGF-I), we analyzed the effect of creatine on the calcineurin, phosphatidylinositol 3 kinase (PI3K) and the mitogen activated-protein kinase (MAPK) pathways. The inhibition of calcineurin by cyclosporin A, the inhibition of extracellular-regulated kinase (ERK) by PD098059 and the inhibition of PI3K by wortmannin did not repress the hypertrophic effect induced by creatine indicating that these pathways are probably not involved. On the other hand, the inhibition of p38 by SB202190 and mammalian target of rapamycin (mTOR) by rapamycin completely inhibited differentiation and creatine did not reverse this effect indicating that the p38 and the mTOR pathways are required for the hypertrophic effect induced by creatine. After 3 days of differentiation, creatine enhanced the phosphorylation state of Akt/PKB, glycogen synthase kinase 3 (GSK3) and 70 kDa ribosomal S6 protein kinase (p70^s6k) (P<0.05). Creatine also affected the phosphorylation state of p38 and the nuclear expression of myocyte enhancer factor 2 (MEF2). This study points out the involvement of the p38 and the Akt/PKB pathways in the hypertrophic effect induced by creatine in differentiating C₂C₁₂ cells.