Measurement of the nuclear DNA content allows classification of human cancers as either diploid or aneuploid. To gain further insight into mechanisms of aneuploidy, we compared the cytogenetic profile of mismatch-repair-deficient diploid versus mismatch-repair-proficient aneuploid colorectal carcinoma cell lines using comparative genomic hybridization and spectral karyotyping. Aneuploid carcinomas revealed an average of 19 chromosomal imbalances per cell line. Such numerical aberrations were exceedingly scarce in the diploid tumors. This pattern of chromosomal aberrations is consistent with a mechanism involving the impairment of chromosome segregation fidelity during mitotic cell division. In support of this idea, we demonstrate the exclusive occurrence of centrosome amplification and instability in all of the aneuploid tumor cell lines analyzed. All diploid tumors contained centrosomes that were functionally and structurally indistinguishable from those in normal human fibroblasts. Due to the observed differences in centrosomes between these two classes of tumors, we incubated the cells with the microtubule depolymerizing drugs nocodazole and griseofulvin. Our results indicate that the aneuploid tumor cell lines have an increased sensitivity to these reagents and a delay in aster formation and microtubule regrowth. However, microtubule nucleation was initiated from one or two centers in both the diploid and aneuploid cells. These observations support the notion that the integrity of the centrosome plays a central role in the development of aneuploidy. Genes Chromosomes Cancer 27:183-190, 2000. Published 2000 Wiley-Liss, Inc.