Deletion of 9p21-22 is a common genetic alteration in dysplastic, in situ, and invasive head and neck squamous cell carcinoma (HNSCC). However, a candidate tumor suppressor gene (TSG) at this site has thus far not been identified in HNSCC. We report homozygous deletion of the recently identified multiple tumor suppressor I (MTSI)/cyclin-dependent kinase-4-inhibitor (CDKN2) gene mapped to 9p21, which encodes the p16 protein, a regulator of cyclin-dependent kinase 4, in six of 16 HNSCC cell lines. We also show absence of the CDKN2 mRNA in all cell lines with CDKN2 deletion as well as in an additional two cell lines without deletion. Overall, we have identified 9p abnormalities in 12 of 16 (75%) cell lines, at least nine of which involved CDKN2. We further demonstrate that the CDKN2 deletion in HNSCC is located within a previously described region of allelic loss between D9S171 and IFNW, which spans a 4 cM region of 9p. However, examination of 36 primary tumors revealed genetic alterations in only seven of 36 (19%) tumors. These results suggest that genetic alterations at CDKN2 are frequent in HNSCC cell lines, but the role of this gene in primary tumors is less compelling. CDKN2 does not appear to be the only TSG on 9p21 in HNSCC, and our results suggest that another region of deletion exists proximal to the IFNW locus.