The role of rodents in the epidemiology of neosporosis was investigated by assaying brain tissue of feral mice (Mus musculus) and rats (Rattus norvegicus) for Neospora caninum. Both mouse and rat brain tissue were extracted for total DNA, and subjected to two different N. caninum-specific nested polymerase chain reaction (PCR) assays. A portion of brain tissue from the mice and rats were also assayed for N. caninum in gerbils or gamma-interferon gene knockout (KO) mice. Of the 105 feral mice tested, 10% were positive in the N. caninum-specific PCR assays. Of the 242 rats tested, 30% were positive in both assays. Although mice and rats had N. caninum by PCR testing, clinical signs of N. caninum infection were not observed nor were N. caninum parasites observed in gerbils or KO mice inoculated with the rodent brain tissue.