In the course of studies to elucidate the relative contribution of simian virus 40 (SV40) large T and small t proteins during oncogenesis, we observed the appearance of pericardial and pleural tumors in 100% of Syrian hamsters injected in the pleural space with wild type SV40. When SV40 was injected via the intracardiac or intraperitoneal routes, more than 50% of hamsters developed mesothelial tumors. Macroscopic, microscopic, ultramicroscopic, and histochemical characteristics identify these neoplasms and derived cell lines as mesotheliomas and mesothelioma-derived cell lines. The SV40 genome was integrated and expressed in the mesotheliomas and derived cell lines. The absence of mesotheliomas in hamsters injected with SV40 small t deletion mutants indicates that the small t protein plays an important role in the development of SV40-induced mesotheliomas. To the best of our knowledge, this is the first definitive report of virus-induced mesotheliomas in mammals.