ATP is well known for its role as an intracellular energy source. However, there is increasing awareness of its role as an extracellular messenger molecule . Although evidence for the presence of receptors for extracellular ATP on skeletal myoblasts was first published in 1983 (Kolb and Wakelam), their physiological function has remained unclear. In this paper we used primary cultures of rat skeletal muscle satellite cells to investigate the role of purinergic signaling in muscle formation. Using immunocytochemistry, RT-PCR, and electrophysiology, we demonstrate that the ionotropic P2X₅ receptor is present on satellite cells and that activation of a P2X receptor inhibits proliferation, stimulates expression of markers of muscle cell differentiation, including myogenin, p21, and myosin heavy chain, and increases the rate of myotube formation. Furthermore, we demonstrate that ATP application results in a significant and rapid increase in the phosphorylation of MAPKs, particularly p38, and that inhibition of p38 activity can prevent the effect of ATP on cell number. These results not only demonstrate the existence of a novel regulator of skeletal muscle differentiation, namely ATP, but also a new role for ionotropic P2X receptors in the control of cell fate.