Many agonists evoke events in the cell nucleus through the control of Ca2+ signals. Recent studies using isolated nuclei have indicated that the nuclear envelope is a store for nuclear Ca2+. However, the release of Ca2+ directly from the nuclear envelope in living cells has never been reported. In the present study, we have investigated the changes of Ca2+ signals at the cyto-nucleoplasmic interface of rat C6 glioma cells using confocal microscopy. Digital imaging indicates that fluo-3, a Ca2+-sensitive fluorescent probe, was concentrated in or around the nuclear envelope. Our experiments also revealed that C6 cells at rest produced spontaneous Ca2+ spikes in the absence of chemical stimulation. The amplitude of the repetitive Ca2+ spikes was higher at the nuclear envelope than in the whole cell or cytosol. After image subtraction, circular rims of Ca2+ release and uptake were seen at the outer boundary of the nucleus. When the cells were treated with thapsigargin (2 muM), a specific Ca2+-ATPase inhibitor, a long-lasting Ca2+ release was observed at the nuclear envelope. Moreover, most of the released Ca2+ was directed inwardly to the nucleoplasm with little outward diffusion. Our results thus indicate: (1) that the nuclear envelope is a Ca2+ store that possesses the ability to discharge and sequestrate Ca2+; and (2) the Ca2+-releasing channels are present in the inner nuclear membrane.