A signaling cascade termed the "spindle checkpoint" monitors interactions between the kinetochores of chromosomes and spindle microtubules to prevent precocious separation of sister chromatids. We have investigated the role of human inhibitor of apoptosis protein (IAP) surviving in regulation of cell division. We demonstrate that HeLa and PtK1 cells transfected or microinjected with surviving anti-sense oligonucleotides produce significantly more polyploid and micronucleated progeny cells and show abortive mitosis when treated with spindle poisons. Furthermore, perturbation of surviving function in HeLa and PtK1 cells with anti-surviving antibodies at the beginning of mitosis affects the normal timing of separation of sister chromatids and disturbs the 3F3/2 phosphoepitope-recognized tension sensing mechanism of the spindle checkpoint. This leads to premature separation of sister chromatids, which results in an uneven distribution of chromosomes between the newly formed progeny cells-an event associated with tumor formation in many cell types. Finally, cells injected with anti-surviving antibody exit mitotic block induced with microtubule drugs. Our data suggest that surviving protein may function within the spindle checkpoint pathway.