The p16INK4A (CDKN2A/MTS1) putative tumor suppressor gene encodes a cyclin dependent kinase inhibitor which plays an important role in the regulation of the G1/S phase cell cycle checkpoint. A high frequency of various p16 gene alterations were consequently observed in many primary tumors. P16 can be inactivated by different mechanisms: i) homozygous deletion, ii) methylation of the promoter region or iii) point mutation. In order to investigate p16 alterations in head and neck cancer (HNC) we analyzed 70 primary tumors of the larynx, pharynx and oral cavity including their corresponding normal mucosa for mutation inactivation by direct sequencing exon 2. We detected only one so far undescribed transversion G to T at position 322 (Asp108Tyr) and a known polymorphism (Ala148Thr) in five cases. The methylation status of the p16 promoter region was analyzed by an improved highly sensitive methylation-specific PCR protocol. P16 methylation inactivation was found in 16 of 55 cases (29%). Our data indicate that p16 point mutations in HNC are less frequent, but inactivation by methylation of the promoter region could be involved in genesis and progression of HNC.