Although genetic alterations of chromosome band 9p21-22 occur frequently in head and neck squamous cell carcinoma (HNSCC) cell lines, alterations of the cyclin-dependent kinase inhibitor p16INK4a located in this region are less common in corresponding primary tumors. To further investigate genetic alterations at 9p21-22 and p16INK4a in primary HNSCC, a paired set of 21 tumors and blood specimens that were shown previously to exhibit allelic loss at 3p and elsewhere, were tested for LOH at 9p21-22 using eight different highly polymorphic marker. Sixteen of the samples (81%) exhibited LOH for at least one marker. Frequent LOH was found surrounding p16INK4a and at three additional non-contiguous regions of 9p21-22. No homozygous deletions were identified. SSCP screening and direct sequence analysis led to the identification of mutations the p16INK4a gene in two tumors. p16INK4a was not hypermethylated in any of the samples studied. Furthermore, there was no correlation between LOH at 9p21-22 and the RB1 tumor suppressor gene. These findings indicate that in the set of tumors that we tested, LOH at 9p21-22 is common in primary HNSCC but that genetic alterations of p16INK4a located in this region are unusual. Additional tumor suppressor genes at 9p21-22 may therefore be involved in the pathogenesis of this tumor.