We performed a retrospective, longitudinal study to determine whether abnormalities in immunohistochemical staining for the epidermal growth factor receptor (EGFR), p53, or cyclin D1 occur before the development of laryngeal carcinoma. Staining was performed on 63 paraffin-embedded biopsies from 18 patients who subsequently developed carcinoma in situ (CIS) or invasive carcinoma of the larynx. These were compared to 71 biopsies from 20 patients who did not develop CIS/cancer (minimum follow-up period, 4 years). Also studied were the 34 biopsies containing CIS and/or carcinoma from those patients who progressed and 22 biopsies obtained concurrently. The two patient groups did not differ significantly in terms of tobacco and alcohol use. Distinct patterns of staining correlated with malignant progression. These included EGFR staining of two thirds or more of the epithelium thickness, a linear basal p53 staining pattern, and strong (3+) staining for cyclin D1 (P < 0.01 for each). These staining patterns also correlated with increasing atypia. In our study population, linear basal staining for p53 and strong staining for cyclin D1 had higher specificity for progression than did EGFR overexpression, which was also seen in association with inflammation and chronic irritation. Marked site-to-site variation was seen in the immunohistochemical staining and in the degree of atypia, suggesting that multiple biopsies are necessary to properly assess risk. These immunohistochemical staining patterns may be clinically useful to predict patients at risk for neoplastic progression.