RNA was isolated from 22 squamous cell carcinomas (SCCs) obtained from diverse sites within the head and neck and from matched normal tissue where available. Tissue samples were then screened for expression of RNA from tumor suppressor gene p16 by utilizing semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis. p16-Specific PCR amplification products generated from tumor samples were subject to further analysis by direct DNA sequencing to determine if any tumor sample harbored a p16 mutation. The results show the presence of mutations in 10 of 22 (45%) of the tumor samples. Mutations comprise two identical point mutations, two small deletions (1 bp and 2 bp), one single-nucleotide insertion, four larger deletions, and an insertion/deletion. No mutations in p16 have been identified by analysis of PCR products generated from normal matched tissue, suggesting that p16 alterations are generated by somatic mutation and are not germline in origin. All 22 samples were analyzed additionally by immunohistochemistry for nuclear expression of the retinoblastoma (RB) tumor suppressor gene product. Results show lack of RB nuclear expression in only one sample, suggesting that mutation of RB is an infrequent event in the development of SCC of the head and neck (SCCHN).