Low-intensity pulsed ultrasound (LIPUS) is commonly used in the treatment of fractures and nonunion-promoting acceleration of healing fractures. In this report, we investigated the implication of the P2 receptors in osteoblast proliferation induced with LIPUS treatment. We observed that ADP, ATP, UTP, and UDP promote osteoblast increase and an increase of intracellular Ca2+, through activation of P2Y receptors. Osteoblasts' expression of the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13 receptors was confirmed. In addition, the participation of the P2Y1 receptor in osteoblast increase and the ADP-dependent increase of Ca2+ concentration were shown. Furthermore, release of ATP/purines was induced by LIPUS treatment. Finally, LIPUS-dependent osteoblast increase was abolished in the presence of the Ca2+ chelator (BAPTA), the inositol 1,4,5-trisphosphate receptor antagonist (2-APB), and the selective P2Y1 receptor antagonist (MRS2179). In conclusion, LIPUS treatment induces osteoblastogenesis via the release of purines, such as ATP, activating P2Y receptors, mainly the P2Y1 receptor. © 2009 Elsevier Inc. All rights reserved.