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The desert tortoise is listed by the United States government as a threatened species in part of its range. A major contributing factor in the decline of this animal has been the presence of an upper respiratory tract disease (URTD) which is characterized by a chronic disease which eventually leads to severe occlusion of the nares with viscous exudate and destruction of the respiratory epithelium. Electron microscopy of infected tissues demonstrated the presence of a mycoplasma-like organism attached to the respiratory surfaces. The mycoplasma was isolated and designated as anew species, with the proposed name Mycoplasma agassizii. The current study was designed to fulfill Koch’s postulates and determine if M. agassizii was the etiologic agent of URTD. Clinically healthy animals with known antibody status were infused intranasally with pooled exudate (n = 8) from ill donor animals, with M. agassizii alone (n = 9) or in combination with Pasteurella testudinis (n = 8), with P. testudinis alone (n = 9), or with sterile broth (n = 12). The pooled exudate was culture positive for M. agassizii. Tortoises which received exudate or M. agassizii alone or in conjunction with P. testudinis were significantly more likely to develop clinical disease (P < 0.0004) than animals which received P. testudinis alone or the broth controls. Tortoises demonstrated a strong immune response to M. agassizii, and seroconversion was seen in all groups with clinical disease. M. agassizii was isolated from the upper respiratory tracts of clinically ill animals up to 6 months postinfection. On the basis of the results of these transmission studies, we conclude that M. agassizii is an etiologic agent of URTD in the desert tortoise.