There are approximately 200,000 new cases of cutaneous squamous cell carcinoma diagnosed each year in the United States, with between 1300 and 2300 deaths per year from metastatic disease. The tumor suppressor p16, encoded by the CDKN2/INK4a locus, has been reported mutated in >or=24% of squamous cell carcinomas. Mutations of the p16 gene have also been found in actinic keratoses, the first identifiable lesion in the continuum from normal skin to squamous cell carcinoma. We hypothesized that there may be an appreciable difference in expression of p16 between normal skin, actinic keratoses, squamous cell carcinoma in situ, and invasive squamous cell carcinoma. Ten actinic keratoses, 10 in situ squamous cell carcinomas, and 10 invasive squamous cell carcinomas were examined using the immunoperoxidase method with antigen retrieval for anti-p16(INK4a) antibody. All 10 actinic keratoses were positive for weak to moderate p16 staining in the lower third to lower half of the epidermis (especially the basal keratinocytes). This staining was significant when compared with the lack of staining seen in normal skin controls. Twenty percent of in situ squamous cell carcinomas had moderate to strong staining in only the lower half to lower two thirds of the epidermis, whereas 70% of the in situ squamous cell carcinomas exhibited full-thickness p16 staining, with no staining in the dermis. Thirty percent of invasive squamous cell carcinomas had full-thickness staining of the in situ component of the lesion, and 100% of invasive squamous cell carcinomas exhibited moderate to strong staining of the invasive component of the lesion. These findings indicate correlation between the increased expression of p16 during the progression of skin from actinic keratosis to in situ squamous cell carcinoma to invasive squamous cell carcinoma. These data may lend further support to the view of the actinic keratosis as a precursor lesion to squamous cell carcinoma.