p53 Protein is a 53-kd nuclear phosphoprotein believed to play an important role in controlling proliferation of neoplastic and normal cells. This "natural tumor suppressor" can be rendered ineffective (or oncogenic) by mutations in the p53 gene or by interactions with proteins synthesized by DNA-transforming viruses, including specific subtypes of human papillomavirus (HPV). We describe the localization of p53 protein in association with HPV in paraffin sections of a spectrum of benign, dysplastic, and malignant anogenital squamous epithelia using immunohistochemical and in situ hybridization techniques. p53 Was detected in 81% of the 48 cases studied. Immunoreactivity for p53 was seen in 83% of the benign and low-grade squamous intraepithelial lesions (SILs), in 73% of the high-grade SILs, and in 86% of the infiltrating squamous carcinomas. In high-grade SILs p53 staining was frequently observed in individual nuclei at various levels of the abnormal epithelium and in the basal layer of the adjacent epithelium, while in squamous metaplasia and low-grade SILs immunostaining for p53 was limited to the basal layer of the epithelium. p53 Was detected in a slightly higher percentage of HPV-positive than HPV-negative epithelia as determined by in situ hybridization. No correlation was observed between p53 immunoreactivity and HPV subtypes. p53 Protein and HPV were detected in anal lesions from a small group of human immunodeficiency virus-positive individuals. Antibodies currently available mainly demonstrate mutant forms of p53 protein that are associated with longer half-lives than the wild-type protein, but demonstration of p53 protein overexpression is not necessarily indicative of malignancy.