Aged male Fischer 344/N rats are prone to developing spontaneous peritoneal mesotheliomas that arise predominantly from the tunica vaginalis of the testes. A definitive cause for the predominance of this neoplasm in F344/N rats is unknown. Investigation of the molecular alterations that occur in spontaneous rat mesotheliomas may provide insight into their pathogenesis as well enable a better understanding regarding the mechanisms underlying chemically induced mesothelioma in rodents. Mesothelial cell function represents a complex interplay of pathways related to host defense mechanisms and maintenance of cellular homeostasis. Global gene expression profiles of spontaneous mesotheliomas from vehicle control male F344/N rats from 2-year National Toxicology Program carcinogenicity bioassays were analyzed to determine the molecular features of these tumors and elucidate tumor-specific gene expression profiles. The resulting gene expression pattern showed that spontaneous mesotheliomas are associated with upregulation of various growth factors, oncogenes, cytokines, pattern recognition response receptors, and pathogen-associated molecular patterns receptors, and the production of reactive oxygen and nitrogen species, as well as downregulation of apoptosis pathways. Alterations in these pathways in turn trigger molecular responses that stimulate cell proliferation and promote tumor survival and progression.