Technical Abstract: Neospora caninum is a major cause of abortion in cattle worldwide. Cattle become infected with N. caninum by ingesting oocysts from the environment or transplacentally from dam to fetus. Experimentally, dogs can act as definitive hosts, but dogs excrete few oocysts after ingesting tissue cysts. A natural definitive host was unknown until now. In the present study, N. caninum was isolated from the feces of a dog. Gerbils (Meriones unguiculatus) fed feces from the dog developed antibodies to N. caninum in the Neospora caninum-agglutination test and tissue cysts were found in their brains. Neospora caninum was isolated in cell culture and in gamma interferon gene knockout mice inoculated with brain homogenates of infected gerbils. The DNA obtained from feces of the dog, from the brains of gerbils fed dog feces, and from organisms isolated in cell cultures inoculated with gerbil brains, was confirmed as N. caninum. The identification of N. caninum oocyst by bioassay and PCR demonstrates that the dog is a natural definitive host for N. caninum.