Toxoplasma gondii, the agent of toxoplasmosis, has a global distribution in a broad spectrum of hosts including mammals and birds. It has been recognized as the major food- and water-borne pathogen in humans due to frequent outbreaks of infections. One-third of the human population worldwide is chronically infected. Infections in healthy pregnant women may cause blindness, intellectual disabilities, or even death of the fetuses. Reactivation of latent infection in immunocompromised patients, such as individuals with AIDS, can cause life- threatening encephalitis. Genetic studies from global samples elucidated high diversity in T. gondii parasites and the isolates vary markedly in virulence. The goal of this proposal is to establish a reference repository and database for natural isolates of T. gondii. From over 1,500 isolates that were previously obtained from different animal hosts and geographical regions in the USDA Animal Parasitic Disease Laboratory, we propose to revive and expand about four hundred representative isolates, and make them available to research community. These diverse parasite isolates are an invaluable resource for future study of transmission, pathogenesis, and evolution of virulence of T. gondii, and provide essential information to treat clinical toxoplasmosis.