The development of head and neck squamous cell carcinoma occurs as a result of the accumulation of genotypic and phenotypic alterations in the upper aerodigestive tract mucosa. Up-regulation of epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor alpha (TGF-alpha), have been identified previously as early events in head and neck carcinogenesis. To determine the timing of increased TGF-alpha and EGFR protein expression in the development of head and neck cancer, we examined progressive mucosal dysplasias from three distinct and complimentary patient groups: (a) samples from patients with lesions demonstrating different degrees of dysplasia (n = 22) compared with mucosa samples from gender and age-matched controls (n = 8); (b) patients with lesions demonstrating different degrees of dysplasia at a single time point (n = 3); and (c) patients who progressed over several years to invasive cancer at the site of dysplasia (n = 7). Immunohistochemical analysis with monoclonal antibodies specific for TGF-alpha and EGFR were used to detect protein expression in all specimens. Protein levels were further quantitated using a computerized image analysis system. In all three groups, we found that TGF-alpha protein levels were elevated in mild dysplasia compared with control normal mucosa and were not further modulated with increasing degrees of dysplasia. In contrast, EGFR levels were relatively low in mild dysplasia and increased with higher degrees of dysplasia. These findings indicate that up-regulation of TGF-alpha and EGFR are distinct events both chronologically and, possibly, mechanistically in the pathogenesis of head and neck squamous cell carcinoma.