In order to investigate the p16 gene alterations in nasopharyngeal carcinoma (NPC), we have examined for mutations and deletions of the p16 gene in samples of NPC including 3 cell lines, 3 xenografts, and 20 primary tumors with matched blood DNA as controls. Using single-strand conformation polymorphism and direct sequencing analysis, no p16 gene mutations were detected in the NPC primary tumors and xenografts. Mutations of the p16 gene were found in three NPC cell lines, but no normal allele was present in these samples. Homozygous deletion of the p16 gene has been identified in 2 (67%) of 3 cases of NPC xenografts and 7 (35%) of 20 cases of primary tumors by comparative multiplex PCR analysis. A homozygous deletion region distal to the p16 locus was observed in a case of NPC primary tumor. Our data document for the first time that alterations of the p16 gene were frequent in NPC and that homozygous deletion was the major mechanism for the inactivation of this gene. These findings suggest that complete inactivation of the p16 gene may play a role in the development of NPC. Moreover, inactivation of other putative tumor suppressor gene(s) outside of the p16 locus within chromosome 9p21-22 may also contribute to the pathogenesis of this disease.