We screened 29 human esophageal squamous cell carcinoma (ESC) cell lines for mutations of the p53 gene through all coding exons and exon-intron junctions. Mutations were found in 22 cell lines (76%), consisting of 20 single-base substitutions, 2 small deletions and 1 single-base insertion. Out of 20 single-base substitution, 5 were located at the exon-intron junctions and mRNAs with abnormal splicing were detected by RT-PCR in 4 of them. A G:C to T:A transversion, which occurred rather frequently in resected tumors of ESC, was observed in only 1 cell line, and, instead, frequent transitions at CpG sites were detected. We also examined 65 fresh tumor materials, from all of which we tried to establish cell lines, and detected mutations in 26 samples (40%). Compared with the results in these fresh tumor materials, the mutation incidence in cell lines was significantly high and the mutation spectrum was also different. From these 65 tumors, 10 cell lines were established, including 3 cell lines from 26 tumors with p53 mutations and 7 cell lines from 39 without mutations, which indicates that there was no significant correlation between the status of the p53 gene in each fresh tumor and its establishment as a cell line. In 7 cell lines established from mutation-free tumors, newly acquired mutations were detected in 5, which suggests that mutations might occur during the process of establishing cell lines.