Oral squamous cell carcinoma cell lines and tumor tissues used for cell line establishment were examined for p53 tumor-suppressor gene mutation using cellular DNAs and RNAs. For sensitive and rapid detection, a newly designed two-stage filtration strategy was used. Full advantage was taken of the single-strand conformation polymorphism (SSCP) of cDNA and cellular DNA fragments amplified by the polymerase chain reaction (PCR), followed by DNA sequencing of mutated fragments. Fourteen out of 15 cells lines and the corresponding five tumor tissues had p53 mutations within the region of exons 5-8. Loss of normal alleles was noted in 14 lines, but not in one in which only mutated transcripts were detected. DNA sequencing indicated six out of 14 mutations to be in positions that have so far not been reported. In two special cases, novel mutations were found in the splicing donor sequence of exon 6, and consequently the cryptic splice site had to be used. Extremely frequent p53 gene mutations indicated that the mutations are likely be intimately involved in the carcinogenesis of oral squamous cell carcinoma.