In the present study, we analyzed p16, retinoblastoma (Rb), and cyclin D1 abnormalities in head and neck squamous cell carcinoma (HNSCC) tissues and cell lines from Korean patients. We found a 40% loss of heterozygosity at the D9S171 locus (9p21 region) these tissues. All eight of the HNSCC cell lines did not express the p16 protein, and in two of these cell lines (Amc-HN-6 and 8), this was due to a deletion of the p16 gene. Three of the cell lines (Amc-HN-3 to 5) that expressed the p16 mRNA had the same nonsense mutation at codon 50 (CGA-Arg to TGA-Ter). The Amc-HN-1 and Amc-HN-7 cell lines, which did not express the p16 mRNA, had a missense mutation at codon 9 (GCC-Ala to GTC-Val) and a silent mutation at codon 106 (CCC-Pro to CCA), respectively. The Amc-HN-2 cell line (p16 exon-positive/mRNA-negative) had a single base deletion at codon 38 (CGG-Arg to CG), which resulted in a frameshift and a consequent stop signal at codon 44. The Rb protein was detected in all of the eight cell lines, although it was inactive in five of these due to hyperphosphorylation. The inverse relationship between p16 and Rb was 62.5% (5/8). Cyclin D1 was overexpressed in all of the eight cell lines. Our results suggest that the abrogation of p16, the overexpression of cyclin D1, and the consequent inactivation of Rb could be important factors in the carcinogenesis of HNSCCs.