Abstract Aneuploidy and chromosome instability (CIN) are hallmarks of solid tumors. Recent studies showed that chromosome missegregation caused by defective kinetochore microtubule attachments during division can give rise to the CIN phenotype. However, the underlying molecular defect that is responsible for the CIN phenotype is poorly understood. On the other hand, increasing evidence suggests that aneuploidy is detrimental to cell growth. Similarly, how CIN cancer cells tolerate a continual state of aneuploidy also remains to be clarified. These are critical questions, given that CIN provides tumor cells a rapid way to adapt to stressful conditions such as chemotherapy. In this study, we examined a panel of ovarian tumor cell lines and report that they exhibit the CIN phenotype. Timelapse movies showed chromosomes in these cell lines were able to align at the spindle equator but exhibited high rates (numbers) of lagging chromosomes, indicative of aberrant kinetochore: microtubule attachments. This observation was confirmed by the finding that a large fraction (numbers) of kinetochores in these cell lines exhibited a variety of attachment defects relative to Hela cells. As the role of Aurora B kinase at kinetochores is to monitor and repair defective microtubule attachments, we quantitated the levels of Aurora B at kinetochores of these ovarian tumor cell lines. We report that Aurora B levels are reduced relative to Hela cells that do not exhibit attachment defects. Consistent with this finding, FRET studies to monitor the real-time Aurora B kinase activity at the kinetochores of two of the three ovarian cell lines showed that kinase activity was weak. This defect was localized to kinetochores, as the overall activity of Aurora B kinase, as monitored by the western blotting of phospho-Histone H3, was similar to that in Hela cells. These findings provide a molecular explanation for kinetochore attachment defects in these ovarian tumor cell lines and, as a consequence, their chromosome instability. We also analyzed the response of these cell lines to treatment with various anti-mitotic agents. We found no obvious growth advantage of these ovarian CIN cell lines in response to drugs, relative to Hela cells. Sensitivity to mitotic death may reflect the variation of the genetic background of a given cell line. Also, the mitotic checkpoint appeared to be required for death in mitosis. Although apoptosis clearly played an important role in mitotic cell death in our studies, other death pathways could participate in this event, when apoptosis was blocked. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3892.