Only 6 autochthonous cases of human Chagas disease have been documented in the U.S.A., however, as many as 5% of immigrants from Latin America may be infected with the etiologic agent, Trypanosoma cruzi. The parasite has been isolated from a variety of wild mammals, particularly in the southeastern region of the U.S.A. The goal of our study was to determine if the sylvatic cycle of T. cruzi infection occurs in Kentucky, and, if present, to assess the prevalence of infection in Warren and Barren counties. Raccoons and opossums were live-trapped between June and December, 2007. Animals were anesthetized with isoflurane, and blood samples were collected using a vacutainer system. Sera were frozen at -80oC for subsequent analysis, and whole blood samples were inoculated, in duplicate, into liver infusion tryptose (LIT) medium and cultured at 27oC. Seventeen T. cruzi isolates from raccoons have been positively identified by hemoculture. A total of 25/44 (57%) raccoon samples were found to be positive by hemoculture or serological analysis. In Warren County 18/25 (72%) of raccoons tested positive for T. cruzi and 7/19 raccoons (37%) in Barren County were positive for the parasite. Eighteen of 43 (42%) of the sera collected from opossums in Warren County and 3 of 5 (60%) from Barren County were judged to be positive by either enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay tests (IFAT). There were no positive hemoculture results for the opossum samples. The infection rates found in the current study for raccoons were slightly higher than those reported in previous studies in the U.S.A. However, the overall prevalence of T. cruzi in opossums (determined by serological analysis) was consistent with previous studies performed in the southeast. To our knowledge, this is the first report of T. cruzi from the state of Kentucky.