p53 alterations were studied in a group of 22 primary squamous cell carcinomas (SCC) of the head and neck and in 10 cell lines derived from SCC. Positive immunohistochemical detection of p53 was accomplished in 10 of 22 primary tumors and in 7 of 10 SCC cell lines. Loss of heterozygosity of chromosome 17p, were the p53 gene is localized, was seen in five of seven SCC lines studied. DNA sequencing of the p53 gene of these five cell lines that had lost one allele showed p53 mutations in the remaining allele. In addition, from six primary SCC that exhibited loss of heterozygosity of chromosome 17p, three showed missense mutations of the p53 gene. The mutations of primary tumors and SCC cell lines were scattered in the midregion of the gene, affecting codons 151, 155, 174, 194, 220, 248, and 273. Five of these mutations modified guanine residues, a phenomenon that has been associated with the effect of carcinogens contained in tobacco smoke. Collectively these data show that approximately 50% of primary tumors and cell lines derived from SCC of the head and neck showed abnormalities of the p53 gene. In addition, it is of interest to note that the most invasive cell lines, as determined in an in vivo assay using xenotransplantation of tumor cells into denuded rat tracheal grafts, exhibited the most intense staining. Similarly, of five very advanced primary tumors, four showed intense p53 immunostain. These observations support the evidence that alterations in this tumor suppressor gene could be related to late events in tumor progression.