A series of 71 surgically resected esophageal squamous cell carcinomas, including 51 cases of formalin-fixed samples and 20 cases of fresh biopsy specimens derived from the high-incidence area of esophageal cancer in China, were systematically analyzed for the presence of human papillomavirus (HPV) infections by light microscopy, electron microscopy (TEM), in situ DNA hybridization, Southern blot hybridization, and polymerase chain reaction (PCR) techniques. On light microscopy, HPV-suggestive lesions were found in a total of 49.0% (25 of 51) of the specimens, including the flat type (22 of 51) and, less frequently, an inverted one (2 of 51). Of the 51 formalin-fixed, paraffin-embedded specimens, 43.1% (22 of 51) contained HPV DNA sequences by in situ hybridization. Of the positive cases, HPV 6 was present in three (5.9%), HPV 11 in three (5.9%), HPV 16 in eight (15.7%), HPV 18 in six (11.8%), double infections with HPV 11/18 in one (2.0%), and HPV 16/18 in one. In most cases the HPV-positive signals were localized in the hyperplastic and/or dysplastic epithelium adjacent to invasive carcinomas. In two specimens, however, HPV DNA sequences were found in the frankly invasive lesions, one being HPV 6 and the other HPV 18. On TEM, HPV-like particles located in the nuclei of koilocytotic cells were demonstrated in two of the five specimens previously shown to be HPV-positive by in situ hybridization. By means of the PCR technique, all specimens positive for HPV by in situ hybridization also contained amplified HPV sequences. Moreover, three additional samples negative by in situ hybridization were found to contain HPV 11 DNA sequences. Of the 20 DNA samples extracted from the fresh carcinoma samples (containing some surrounding tissues as well) 9 were shown to contain HPV DNA sequences by Southern blot hybridization under low-stringency conditions. Of these, eight samples remained positive when hybridized with the probe cocktail of HPV 11, 16, 18, and 30 DNA under high-stringency conditions. HPV DNA sequences in these carcinoma specimens appeared to be present mainly in an integrated form. The present results confirm the HPV involvement in esophageal squamous cell lesions and suggest that HPV infection might be an important etiologic factor in the pathogenesis of esophageal cancer, most probably acting synergistically with other carcinogenic factors.