In human osteoblast-like MG-63 cells, extracellular ATP increased [³H]thymidine incorporation and cell proliferation and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced [³H]thymidine incorporation. ATP-induced [³H]thymidine incorporation was mimicked by the nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptor antagonist reactive blue 2 did not affect [³H]thymidine incorporation, whereas the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced [³H]thymidine incorporation, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X₄, P2X₅, P2X₆, and P2X₇, but not P2X₁, P2X₂, and P2X₃, receptors. In fura 2-loaded cells, not only ATP, but also UTP, increased intracellular Ca²⁺ concentration, and inhibitors for several Ca²⁺-activated protein kinases had no effect on ATP-induced DNA synthesis, suggesting that an increase in intracellular Ca²⁺ concentration is not indispensable for ATP-induced DNA synthesis. ATP increased mitogen-activated protein kinase activity in a Ca²⁺-independent manner and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced kinase activity. Furthermore, the mitogen-activated protein kinase kinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors by activating a mitogen-activated protein kinase pathway.