The mammalian securin, pituitary tumor-transforming gene (PTTG), is overexpressed in several tumors and transforms cells in vitro and in vivo. To test the hypothesis that PTTG overexpression causes aneuploidy, enhanced green fluorescent protein (EGFP)-tagged PTTG (PTTG-EGFP) was expressed in human H1299 cancer cells (with undetectable endogenous PTTG expression) and mitosis of individual live cells observed. Untransfected cells and cells expressing EGFP alone exhibited appropriate mitosis. PTTG-EGFP markedly prolonged prophase and metaphase, indicating that PTTG blocks progression of mitosis to anaphase. In cells that underwent apparently normal mitosis (35 of 65 cells), PTTG-EGFP was degraded about 1 min before anaphase onset. Cells that failed to degrade PTTG-EGFP exhibited asymmetrical cytokinesis without chromosome segregation (18 of 65 cells) or chromosome decondensation without cytokinesis (9 of 65 cells), resulting in appearance of a macronucleus. Fifty-one of 55 cells expressing a nondegradable mutant PTTG exhibited asymmetrical cytokinesis without chromosome segregation, and some (4 of 55) decondensed chromosomes, both resulting in macronuclear formation. During this abnormal cytokinesis, all chromosomes and spindles and both centrosomes moved to one daughter cell, suggesting potential chaos in the subsequent mitosis. In conclusion, failure of PTTG degradation or enhanced PTTG accumulation, as a consequence of overexpression, inhibits mitosis progression and chromosome segregation but does not directly affect cytokinesis, resulting in aneuploidy. These results demonstrate that PTTG induces aneuploidy in single, live, human cancer cells.