We studied 11 head and neck squamous carcinoma (HNSC) cell lines and 46 primary tumors for p16 gene status by protein, mRNA, and DNA genetic/epigenetic analyses to determine the incidence, the mechanism(s), and the potential biological significance of its inactivation. Of the 11 cell lines, only 1 showed intact p16 and 10 lacked its protein and mRNA; DNA analysis of these 10 cell lines showed 2 homozygous deletions, 6 methylations at exon 1 and 2, and 2 with no detectable abnormalities. In primary tumors, 16 (34.7%) of the 46 showed detectable p16 protein and mRNA; of these, 12 had no DNA abnormalities and 4 had only exon 2 methylation. Loss of p16 expression was found in three tumors with concurrent mutation at exon 2 and methylation at exon 2 (two) and both 1 and 2 (one). Of the 30 tumors that lacked p16 protein, 27 also lacked mRNA, 1 had detectable p16 mRNA, and 2 failed RT-PCR amplification. Twenty-two of the thirty tumors showed DNA alterations and eight manifested no abnormalities; DNA alterations comprised 6 homozygous deletions, 2 concurrent mutations and methylation of exon 2, and 13 with methylation at exon 1 and exons 1 and 2 (12 with methylation only and 1 with mutation) at exon 1. Except for patients' gender (P = 0.02), no significant correlation between p16 and clinicopathological factors was observed. We conclude that in HNSC 1) intragenic p16 alterations are infrequent events, 2) methylation of exon 1 constitutes a common mechanism in silencing the p16 gene, 3) p16 inactivation may play an important role in the early development and progression of HNSC, and 4) no association between p16 alterations and conventional clinicopathological factors was noted in this cohort.