In addition to T cell leukemias and lymphomas, human T cell leukemia virus type 1 (HTLV-1) infection has been associated with nonhematologic malignancies and described as the cause of one case of small-cell lung carcinoma. Infected primary epithelial cells have been isolated from sweat gland and oral mucosae of HTLV-1-infected human patients. In the present study, epithelial neoplasms developed in two rabbits experimentally infected with a molecular clone of HTLV-1 (strain K30p). Serologic detection of anti-HTLV-1 and isolation of virus from blood lymphocytes at multiple time points postinjection established a course of chronic asymptomatic infection in both. One rabbit, infected for 5.5 years after intramuscular injection of HTLV-1 DNA, developed a thymoma having features of medullary differentiation. HTLV-1 provirus was detected in both thymocytes and neoplastic epithelium isolated discretely from the thymoma by laser capture microdissection. These findings provide the first experimental evidence of HTLV-1 disease after infection by HTLV-1 DNA injection. Endometrial adenocarcinoma occurred in a second rabbit 2.5 years after its inoculation with cell-associated virus. In this second case, an epithelial cell line derived ex vivo from a metastatic lesion produced virus in culture. In tumors from each of the two rabbits, the neoplastic epithelium was infected and harbored monoclonally integrated HTLV-1 provirus. Although monoclonal provirus integration alone does not establish retroviral cause of carcinogenesis unequivocally, these and other accumulating data indicate that there may be a role for HTLV-1 in diseases associated with infection of epithelia, including some epithelial cancers.