The 18q chromosomal region is frequently lost in head and neck squamous cell carcinomas (HNSCCs). Several candidate tumor suppressor genes have been mapped to this chromosomal region, including DCC, DPC4, and MADR2. The latter two genes are members of the Smad family, key downstream mediators in the transforming growth factor beta signaling pathway, and their alterations could confer resistance to transforming growth factor beta and contribute to tumorigenesis. Nevertheless, genetic alterations of DCC and DPC4 in HNSCC have not been frequently reported. To further investigate the extent and significance of the loss of the 18q chromosomal region in HNSCC, we performed detailed mapping at this region in a set of 50 primary HNSCCs using 19 highly polymorphic microsatellite markers. We detected loss of heterozygosity in 84% of the tumors tested and were able to identify three minimal deleted regions encompassing markers D18S467-D18S474 at 18q12 (4 cM), D18S1099-D18S487 at 18q21.1 (3 cM), and D18S69-41 at 18q21.1-q21.2 (2 cM). Of these minimal deleted regions, only one harbors a known candidate tumor suppressor gene, DCC, which maps telomeric to D18S46. In addition, the role of the MADR2 gene in HNSCCs was investigated by examining nine HNSCC cell lines for alterations of the gene by reverse transcription-PCR and direct sequencing analysis. No mutations or polymorphisms were detected, making this gene an unlikely target of the frequent loss at 18q in HNSCC. Our data indicate high frequency of loss of heterozygosity at 18q in HNSCC and the presence of at least two as yet unidentified tumor suppressor genes in this chromosomal region. Additional efforts to identify these putative tumor suppressor genes are warranted.