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Background: Toxoplasma gondii (T. gondii) has been associated with acute food-borne illness, chronic low-grade inflammation, neuropsychiatric conditions and reactivation of chronic latent infection in immunocompetent hosts. Primary infection with T. gondii in pregnant women can lead to congenital toxoplasmosis. In addition to well-known oral tissue-cyst or oocyst ingestion, we hypothesized that the very high prevalence of T. gondii in certain populations exposed to agricultural dust could be, in part, a consequence of airborne infection with oocysts. Methods: We collected environmental dust samples from an area with a reportedly high T. gondii seroprevalence in the Old Order Amish population, in Lancaster, Pennsylvania. Samples included: a) air filters from air-conditioning units; b) swabs of settled dust; and c) vacuum filters containing airborne field dust. Pools of the swabs and shredded sub-samples of the air filters were fed to pigs, with inoculation into mice of heart tissue from seroconverted pigs. We also investigated the presence of T. gondii DNA using PCR amplification. Results: Only one pig seroconverted. However, bioassay of pig heart tissue further inoculated into mice showed no evidence of T. gondii infection. Consistently, no evidence of T. gondii DNA was revealed in any sample. Conclusions: No evidence of airborne transmission was found in the environmental samples that were examined.