2,2'-dithiodipyridine (2,2'-DTDP), a reactive disulphide that mobilizes Ca 2 + in muscle, induced an increase in cytoplasmic free Ca 2 + concentrations ([Ca 2 + ] i ) in MG63 human osteosarcoma cells loaded with the Ca 2 + -sensitive dye fura-2. 2,2'-DTDP acted in a concentration-independent manner with an EC 5 0 of 50 μM. The Ca 2 + signal comprised an initial spike and a prolonged increase. Removing extracellular Ca 2 + did not alter the Ca 2 + signal, suggesting that the Ca 2 + signal was due to store Ca 2 + release. In Ca 2 + -free medium, the 2,2'-DTDP-induced [Ca 2 + ] i increase was not changed by depleting store Ca 2 + with 50 μM bredfeldin A (a Golgi apparatus permeabilizer), 2 μM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), 1 μM thapsigargin (an endoplasmic reticulum Ca 2 + pump inhibitor) or 5 μM ryanodine. Conversely, 2,2'-DTDP pretreatment abolished CCCP and thapsigargin-induced [Ca 2 + ] i increases. 2,2'-DTDP-induced Ca 2 + signals in Ca 2 + -containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 2,2'-DTDP-induced Ca 2 + release was inhibited by a thiol-selective reducing reagent, dithiothreitol (5-25 μM) in a concentration-dependent manner. Collectively, this study shows that 2,2'-DTDP induced [Ca 2 + ] i increases in human osteosarcoma cells via releasing store Ca 2 + from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The 2,2'-DTDP-induced store Ca 2 + release appeared to be dependent on oxidation of membranes.