Chromosome painting by fluorescence in situ hybridization (FISH) was used to examine abnormalities identified by G-banding in colon cancer lines, DLD-1 (ATCC CCL 221) and HCT-15 (CCL 225). DNA libraries from chromosomes comprising these abnormalities (i.e., N2, N8, N11, N16, N17 and N20) were used to prepare paint probes by PCR amplification. Of these paint probes, N2 and N8 exhibited additional chromosome-specific hybridization signals on centromeres that were also useful as a marker for chromosome identification. Results from FISH-painting and G-band analysis were consistent and permitted our quantitative analysis on karyotype evolution in vitro. In DLD-1, predominant cells having trisomic N20 in early passages were replaced by others with disomic N20 in late passages resulting in the trisomic 2p13-23 segment as the only deviation from the diploid content. In HCT-15, predominant cells having t(16;16) and double Y chromosome copies in early passages were replaced by those bearing the paired N16 and single Y chromosome in later passages. Thus cultures changed from the predominant hyperdiploidy to the sole pseudodiploidy with increased number of normal chromosomes.