A key step in mitosis is the sister-chromatid separation at the metaphase-anaphase (M/A) transition. Several earlier studies had suggested that Ca2+ signal is involved in regulating this process in somatic cells. The detailed mechanisms, however, are not yet well understood, In this study, we used the GFP-gene fusion method and a living-cell imaging technique to examine the effects of suppressing cytosolic Ca2+ level on the mitotic process in HeLa and PtK2 cells. We observed that application of the Ca2+ chelator BAPTA/AM can block or severely delay the M/A transition. This blockage was caused by a failure in activating the anaphase-promoting complex (APC), since both cyclin B and securin could not be degraded under this situation. Furthermore, using YFP-labeled tubulin, we found that the mitotic spindle structure in most of the BAPTA-treated cells gradually deformed with time. Other Ca2+ signal blockers, such as heparin, also produced a similar effect. These results suggest that one pathway for the blockage of M/A transition by suppressing cytosolic Ca2+ level is due to its interference with the mitotic spindle checkpoint. (C) 2003 Elsevier Science (USA). All rights reserved.