Chemical induction of premature chromosome condensation (PCC) was investigated and optimized to be able to analyze the chromosomal constitution of cancer cells independent of mitosis and with minimal culture artifacts. A potent protein phosphatase inhibitor, calyculin A, was used to induce PCC in normal diploid cells, in several established human tumor cell lines, and in cells isolated from freshly dissected adenomatous polyps of a patient with hereditary colorectal cancer. In parallel, mitotic arrest was pursued by use of Colcemid. In cell lines, a difference of up to 10-fold was found between frequency of cells with PCC induced by calyculin A (PCC index) and the mitotic index after treatment with Colcemid. In the fresh tumor specimens, Colcemid failed to result in metaphase formation, whereas a regimen of 80 nM calyculin A for 75 min, after only 2 days of culturing, resulted in a PCC index of 2-5%. pq-COBRA-FISH (COmbined Binary RAtio labeling-fluorescence in situ hybridization) was used for a detailed analysis of four cell lines treated with calyculin A, which proved that PCC spreads are amenable to molecular karyotyping, and a comparison between PCC spreads and metaphases from mitotic arrest revealed no discrepancies in karyotypes. pq-COBRA-FISH on PCC spreads from fresh colon tumor samples revealed only numerical and no structural abnormalities. Calyculin A-induced PCC combined with multicolor FISH gives a new opportunity for analysis of the chromosomal constitution of G1 and G2 cancer cells and may find application in the study of the role of chromosome instability in cancer development.