From July 1999 to July 2000, 40 patients from the Surgical Oncology and Gastroenterology Departments of Irmandade da Santa Casa de Misericórdia de Porto Alegre were enrolled in a prospective study in order to find the frequency of human papillomavirus DNA (HPV-DNA) in esophageal squamous cell carcinoma. They were compared to 10 normal individuals (controls). Digene Inc. (Digene Corporation, Gaithersburg MD, USA) Hybrid Capture II assay, a chemoluminescent method, was used in both groups. The samples were collected from the tumor group by endoscopic biopsy in 20 cases, and by surgical specimens in the other 20 cases; the control group all underwent endoscopic biopsies. The cut-off point in the analytical assay for HPV-DNA was 1.0 pg/mL. Digene Corp. Hybrid Capture II assay tests HPV-DNA and differentiates high and low-risk for malignization virus groups. Gender, age, location of the tumor in esophagus and degree of differentiation on histology were also analyzed. There was one positive case (1.14 pg/mL), a 2.5% frequency of low-risk HPV-DNA type in the esophageal squamous cell carcinoma group (CI 95%: 0.00-7.34%). This group of patients consisted of 30 male and 10 female individuals, similar to the distribution seen in large series of such patients from the Brazilian population. The mean age was 63 years. Endoscopic samples of 10 patients who underwent endoscopic biopsy procedure for aleatory reasons, with normal looking esophageal mucosa, were tested to detect HPV-DNA, as controls. There was one positive case (1.17 pg/mL) of high-risk HPV-DNA type, resulting in a 10% positive rate. HPV-DNA is rarely found in esophageal squamous cell carcinomas: we found a 2.5% frequency, with confidence interval of 0-7.34%; and this is in the same range of controls (10% frequency, with CI: 9.1-10.9%). Both patients had a viral load quite near the 1.0 pg/mL cut-off point.