The possible etiological role of human papillomavirus (HPV) in esophageal carcinogenesis was evaluated in Alaska Natives in whom the incidence of esophageal cancer is 1.3 and 3.8 times higher than in US Caucasian men and women, respectively. Fixed paraffin-embedded esophageal tissues from 32 cases of squamous-cell carcinoma (SCC) and 3 cases of adenocarcinoma (AC) diagnosed between 1957 and 1988 were analyzed by polymerase chain reaction (PCR) and in situ hybridization for HPV DNA sequences. Detection of the human beta-globin gene by PCR was used as a control for sufficiency of DNA and its potential for amplification in the tissue samples. Twenty-five of the tumor tissues were considered adequate for PCR analyses; HPV DNA was detected in 10 of 22 SCCs and was not found in 3 ACs. Seven of the 10 HPV-positive tissues contained sequences from the E6 gene of HPV type 16. Koilocytosis, an epithelial change consistent with HPV infection, was found in 80% of the esophageal squamous-cell tumors with HPV DNA and in 75% of those without HPV DNA. The detection of amplifiable cellular DNA was related to recentness of diagnosis; however, the detection of HPV DNA within amplifiable specimens was not related to recentness of diagnosis. A 413-bp sequence from the L1 open reading frame of HPV 16 from esophageal tissue of 2 patients was identical to sequences previously identified in cervical cells from other Alaska Natives. Our results provide molecular evidence of HPV infection, especially type 16, in archival esophageal cancer tissues from 45% of those patients whose specimens contain adequate DNA for PCR analysis.