BACKGORUND: The physiological activity of osteoblsts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+ ]i) in osteoblasts. The cellular regulation of ([Ca2+ ]i) in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or as toxins in cells. METHODS: Osteoblasts were isolated from the femurs and tibias of neonatal Sprague-Dawley rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+ -sensitive fluorescent dye, Fura-2 AM ester, and fluorescence images were monitored using a cooled CCD camera. Ca-spike changes upon ATP application were checked for (1) osteoblasts in Ca2+ -free and 2.5 mM CaCl2 normal Tyrode solution, (2) osteoblasts in which the Ca2+ of the endoplastic reticulumin had been depleted with ryanodine, thapsigargin ord caffein, and (3) osteoblasts pretreated with H2O2, in which the expression of iP3 receptor was checked by Western blotting. RESULTS: ATP increased intracellular free Ca2+ regardless of extracellular Ca2+ concentration. When the intracellular Ca2+ store was depleted, the level of increased Ca2+ activity by ATP was suppressed. H2O2 sustained the Ca2+ increase induced by ATP. The expression of iP3 receptor was enhanced by H2O2. CONCLUSiONS:H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.