Anal carcinomas were tested for the presence of human papillomavirus (HPV) RNA transcripts by in situ hybridization using 125I-labeled RNA probes. The HPV transcripts were detected in 73.2% of 41 nonglandular anal carcinomas but in none of six anal or 11 rectal adenocarcinomas. In anal nonglandular carcinomas, no difference in the percentage of tumors that were HPV RNA positive was observed between tumors classed as basaloid carcinomas or squamous cell carcinomas. Patients with HPV RNA-negative tumors were significantly older (9.2 years) than those with HPV RNA-positive tumors. Anal intraepithelial neoplasia Grade 3 in the surrounding epithelium was seen in 14 of 25 assessable HPV RNA-positive tumors and in none of nine assessable HPV RNA-negative tumors. Anal tumors may include two separate clinical and biologic groups distinguished by their dependence, or lack of dependence, on HPV RNA expression for maintenance of the neoplastic state.