The receptor for advanced glycation end products (RAGE), a multiligand receptor of the immunoglobulin superfamily, has been implicated in the inflammatory response, diabetic angiopathy and neuropathy, neurodegeneration, cell migration, tumor growth, neuroprotection, and neuronal differentiation. We show here that (i) RAGE is expressed in skeletal muscle tissue and its expression is developmentally regulated and (ii) RAGE engagement by amphoterin (HMGB1), a RAGE ligand, in rat L6 myoblasts results in stimulation of myogenic differentiation via activation of p38 mitogen-activated protein kinase (MAPK), up-regulation of myogenin and myosin heavy chain expression, and induction of muscle creatine kinase. No such effects were detected in myoblasts transfected with a RAGE mutant lacking the transducing domain or myoblasts transfected with a constitutively inactive form of the p38 MAPK upstream kinase, MAPK kinase 6, Cdc42, or Rac-1. Moreover, amphoterin counteracted the antimyogenic activity of the Ca 2؉ -modulated protein S100B, which was reported to inhibit myogenic differentiation via inactivation of p38 MAPK, and basic fibroblast growth factor (bFGF), a known inhibitor of myogenic differentiation, in a manner that was inversely related to the S100B or bFGF concentration and directly related to the extent of RAGE expression. These data suggest that RAGE and amphoterin might play an important role in myogenesis, accelerating myogenic differentiation via Cdc42-Rac-1-MAPK kinase 6-p38 MAPK.Myogenesis is a multistep process in which myoblasts cease to proliferate, express genes responsible for differentiation, and fuse into multinucleated cells, the myotubes, which finally build up the myofibrils (1,2,18,31,33,39,59). Several extracellular factors have been identified that participate in the regulation of myogenesis, some of which promote myoblast differentiation and/or myotube formation, while other factors inhibit these processes. Insulin, insulin-like growth factors (IGF I and IGF II), neuregulin, and nerve growth factor belong to the first category of agents (13-15, 28, 45), while tumor necrosis factor alpha (TNF-␣), basic fibroblast growth factor (bFGF), and transforming growth factor  belong to the second category (12,29,30,35,37,40,42,50,56). However, IGF I and IGF II were reported to promote or inhibit myogenic differentiation depending on the absence or presence of TNF-␣, respectively (16), and down-regulation of nerve growth factor low-affinity receptor was shown to be required for myoblast terminal differentiation (12). Signaling pathways implicated in the transduction of the effects of these agents acting on myoblasts include (i) the mitogen-activated protein (MAP) kinase (MAPK) p38 and Akt, the activation of which is required for myogenesis (5,9,10,17,32,44,55,57,62,66); (ii) an NF-B-dependent pathway activated by cytokines such as TNF-␣, which interferes with myogenesis (30); (iii) a PW1-dependent, NF-Bindependent activation of caspases in the absence of apoptosis (8); (iv) the Ras-MEK-extracellular sign...