It is well established that cellular Ca2+ is an important messenger that controls many nuclear functions but the source of nuclear Ca2+ is far from clear. It has long been thought that Ca2+ is translocated from the cytosol over a long distance to activate the nuclear transcription machinery. However, this model is at best an incomplete one. With the aid of confocal microscopy, we observed tubules extended deep inside the nucleus of C6 cells in agreement with previous studies (Fricker et al. (1997) J. Cell Biol. 136, 531-544). When cells were stimulated with phorbol 12-myristate 13-acetate or phorbol 12,13-diacetate, Ca2+ was released from these tubules. DiOC6(3), a vital marker for intracellular membranes, stained the tubule in the nucleus of the same cell used for Ca2+ imaging. Moreover, results from labelling the cells with rhodamine 123 further indicate that the tubule was formed by a double-membraned invagination with mitochondria inside. Studies with acridine orange showed that chromatin was excluded from the tubules. Taken together, our results demonstrate that the nuclear tubule is a structural entity responsible for the release of Ca2+ into the nucleoplasm after stimulation with phorbol ester.