It is widely believed that most human tumors, including esophageal squamous cell carcinoma (ESCC), arise through multistep genetic and cytogenetic alterations. The time sequence of these alterations, however, is still unknown. The present study was designed to differentiate common early changes from uncommon later ones with combined comparative genomic hybridization (CGH) and ploidy analyses in multiple or single samples of 12 ESCCs. We first demonstrated that the mean copy numbers of chromosomes 3 and 11, determined directly by fluorescence in situ hybridization, showed linear correlation with the mean copy numbers calculated from the G/R ratio of CGH and DNA ploidy (R(2)=0.714, P<0.0001). On this basis, we estimated the absolute copy numbers of chromosomal parts by applying the ploidy-dependent threshold criteria to the G/R ratio data after the criteria were corrected by the percentage of tumor cells in each sample. One-copy changes in the DNA-diploid stage may give large shifts of the G/R ratio, even after tetraploidization, whereas those after tetraploidization undergo small shift. Using the tumors with multiple samples, it was actually demonstrated that most of the gains common to the samples in individual tumors showed the large shifts. Though early changes varied from tumor to tumor in the nine informative cases, it was found that gains of 3q (5/7: number of cases with large-shift 3q+/total number of cases with 3q+), 8q (3/4), 11q13 (4/5), and 14q (3/4) were early events, while losses of 3p (2/8), 5q (1/5), 13q (1/5), and 21q (1/5), and gains of 1p (1/4) and Xq (1/4) were later events in progression of individual tumors.