Primary squamous cell carcinoma (SCC) of the colorectum is an extremely rare malignancy of unknown etiology and pathogenesis. We describe an 87-year-old man with primary SCC of the rectum. Routine histology demonstrated a squamous metaplasia-dysplasia sequence of the rectal mucosa with subsequent malignant transformation. Molecular biologic analysis using polymerase chain reaction (PCR) and in situ hybridization revealed the presence of human papillomavirus type 16 (HPV-16) DNA within metaplastic, dysplastic, and SCC lesions and in tumor-free rectal mucosa. Moreover, nested reverse-transcription PCR showed transcriptional activity of the viral E6/E7 oncogenes in tumor tissue and tumor-free rectal mucosa. By contrast, 4 typical adenocarcinomas of the rectum and their adjacent normal mucosa were found to be negative for HPV by nested PCR. In line with the well-established concept of HPV-associated anogenital carcinogenesis, our results strongly suggest an etiologic role of HPV-16 in the pathogenesis of the metaplasia-dysplasia-SCC sequence in the case described.