The progression potential of preinvasive epithelial lesions is usually evaluated by assessing the degree of histologic dysplasia. We examined p16, retinoblastoma protein (pRb), and proliferating cell nuclear antigen (PCNA) immunophenotypes in 57 cases of previously untreated squamous cell carcinoma (SCC) of the upper digestive tract and in the neighboring normal and dysplastic epithelia. Tissue samples were examined for homozygous deletion of exon 2 of the p16 gene using polymerase chain reaction (PCR) analysis. The PCNA index increased with increasing grade of dysplasia. The pRb protein was expressed in 89% of the samples of SCCs and in the neighboring dysplasias and carcinoma in situ (CIS). In cases with a lack of pRb expression, corresponding preinvasive lesions were also negative. Lack of p16 expression was found in 82% of SCCs. The prevalence of p16 expression decreased with increasing grade of dysplasia. Molecular analysis of the p16 gene showed homozygous deletion in 37% of SCCs, 33% of CIS, and 15% of the samples of normal epithelia. Our data indicate that inactivation of p16 may play an important role in early head and neck carcinogenesis, whereas the mutation of Rb may be an infrequent event. The p16 immunophenotype might be a biomarker for an increased risk of progression in squamous dysplasia.