Many human tumors contain variant cells that, unlike their normal counterparts, possess indefinite proliferative potential in vitro. However, little is known of the relevance of these immortal cells to human carcinomas in vivo. To investigate immortality in a human tumor system, we established cultures from different stages of head and neck squamous carcinoma (SCC-HN). All the neoplastic cultures were transformed because they showed very low cornification in surface or suspension culture and were partially or completely resistant to suspension-induced death. Immortal variants were not detected in premalignant erythroplakia cultures, but their frequency increased with tumor progression, indicating that immortality is a late event in carcinogenesis. Some late-stage carcinomas still produced senescent cultures, but, significantly, all recurrent tumors were immortal. Immortal but not senescent carcinoma cultures were associated with p53 dysfunction and a high frequency of allele loss, indicative of tumor suppressor gene inactivation. These results show that there are at least two classes of human SCC-HN that are phenotypically and genotypically distinct and that the pathological stage of a given tumor is not necessarily indicative of the kind of cells it contains.