Trypanosoma cruzi, causative agent of Chagas disease, is commonly detected in wildlife in North America. Twelve species of mammals from six states were tested for antibodies to T. cruzi using indirect immunofluorescent antibody testing. Culture attempts were conducted on animals from Georgia and Florida. In general, the highest antibody prevalence rates were found in raccoons (Procyon lotor) (33-68%), followed by Virginia opossums (Didelphis virginiana) (28-52%), but antibodies were also detected in small numbers of other animals. Culture-based prevalence rates for raccoons were greater than those for opossums; however, antibody prevalences of raccoons and opossums were not different, indicating similar exposure levels. Several raccoon and opossum population parameters were examined with regards to prevalence and results indicated that T. cruzi prevalence varied by host species, host characteristics, and geographic region. The results of this study provide data to guide future studies on the natural history of T. cruzi in the United States.