To identify the stage in head and neck carcinogenesis at which p53 abnormalities become important, we evaluated 512 squamous epithelial tissue samples (201 pre-invasive and 209 invasive lesions, as well as 102 normal epithelia) in specimens from two institutions. Of 311 patients evaluated, 128 did not have an invasive carcinoma. The frequency of p53 overexpression in the pre-invasive lesions was not influenced by an adjacent invasive tumor. There was an increasing frequency of p53 overexpression in specimens from histologically normal epithelium (5%, with only scattered basal layers positive) to mild (28%) and moderate dysplasia (47%). p53 overexpression was also significantly more common in lesions with severe dysplasia (54%) and carcinoma in situ (CIS; 50%) than in those with mild dysplasia. To further evaluate the timing of p53 alterations in the development of head and neck cancer, paired epithelial samples (one pre-invasive and one invasive) from the same patient were evaluated. A discordance in the p53 staining pattern was seen in approximately one-third of the cases. When a discordance was present, the frequency with which the invasive lesion was positive but the non-invasive negative decreased from 97% (invasive versus normal epithelium) to 50% (invasive versus severe dysplasia or CIS). When p53 expression was evaluated by site, buccal lesions appeared to overexpress p53 more frequently than tongue lesions. In conclusion, p53 protein overexpression is an early event in head and neck carcinogenesis and may represent a biomarker for patients with pre-invasive lesions.