Anal canal carcinomas account for between 1% and 2% of all gastrointestinal carcinomas in the United States. By far, the most common carcinoma in this site is squamous cell carcinoma, but the differential diagnosis typically includes poorly differentiated adenocarcinoma and well-differentiated neuroendocrine carcinoma or carcinoid tumor. Because the first diagnostic specimen received in the pathology laboratory is usually a small, sometimes suboptimal biopsy, the distinction of these types of carcinoma can be difficult. However, accurate diagnosis is imperative, because the treatment differs between squamous carcinoma (chemoradiation) and the other types of carcinoma (surgical therapy). The p63 protein has been previously shown to be involved in epithelial proliferation and differentiation, and is known to be related to squamous carcinomas in many sites. Therefore, we undertook to ascertain its usefulness in the diagnosis of squamous carcinomas in the anal canal. We retrieved 24 anal squamous carcinomas, 68 colorectal adenocarcinomas (including a tissue microarray), and 32 colorectal neuroendocrine carcinomas from the archives at the University of Michigan, and immunostained them for the p63 antigen. As a result, this immunohistochemical stain had a specificity of 98% and a positive predictive value of 92% for squamous cell carcinoma once invasive carcinoma had been established. It also stained the dysplastic epithelial cells in adjacent areas of anal intraepithelial neoplasia. We report that the p63 immunostain is a highly specific and useful tool in the diagnosis of carcinomas of the anal canal.