The expression of human leukocyte antigen (HLA) class I molecules on the cell surface is necessary for the presentation of peptide antigens to cytotoxic CD8+ T lymphocytes of the immune system. Down-regulation of HLA class I gene expression has been implicated in tumorigenesis, including squamous cell carcinoma of the head and neck (SCCHN). Loss of MHC class I antigens may be one mechanism by which tumor cells escape immune detection. We performed prospective immunostaining of 26 primary SCCHN tumors and samples of normal mucosa harvested several centimeters away from the primary tumor, using a large panel of antibodies directed against allele-specific as well as monomorphic determinants of HLA class I molecules. Loss of expression of HLA class I proteins in the tumor was found in 50% (13 of 26) of primary tumors and was highly correlated with HLA loss in the corresponding normal mucosa (P < 0.0001). Further analysis demonstrated that the loss of HLA class I expression in the tumor was significantly associated with regional lymph node metastases (nodal stage; P = 0.0388), and that the number of HLA class I alleles lost in the normal mucosa was associated with subsequent development of a new primary aerodigestive tract cancer (P = 0.042). A patient with two metachronous cancers available for analysis had no evidence of HLA loss in the first tumor, demonstrated allelic loss in the second cancer, and subsequently died of disease. These results suggest that the loss of expression of HLA class I alleles may have prognostic implications.