In order to define more clearly the role of chromosome 9 loss in head and neck squamous cell carcinoma (HNSCC), 29 invasive carcinomas and 17 preinvasive lesions were analyzed for loss of heterozygosity (LOH) on chromosome 9. We found LOH in 21 of 29 (72%) HNSCC tumors using highly polymorphic microsatellite markers. In 17 of 21, LOH was found at all informative sites on the p arm with no LOH of the q arm. Further mapping in tumors, with partial LOH of the 9p arm, localized a common region of loss between markers D9S165 and D9S156. Deletion of this region on chromosome 9 has been found in several other tumor types implying the presence of a tumor suppressor gene at this locus. The inactivation of a tumor suppressor gene on chromosome 9p may represent the most commonly described genetic alteration in HNSCC. A similar incidence of allelic loss on chromosome 9p was identified in 12 of 17 (71%) preinvasive lesions. The identical frequency of loss in preinvasive and invasive lesions suggests that loss of 9p is an early event in HNSCC progression.