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Gopher tortoise (Gopherus polyphemus) populations on four tracts of public lands in northern and central Florida were studied from 1998 to 2001 to assess the effects of mycoplasmal upper respiratory tract disease (URTD) Adult gopher tortoises (n = 205) were marked for identification, serum and nasal flush samples were obtained for mycoplasmal diagnostic assays, and clinical signs of URTD (nasal discharge, ocular discharge, palpebral edema, and conjunctivitis) were evaluated A subset or tortoises (n = 68) was radio-instrumented to facilitate repeated sampling and document potential mortality Presence of serum antibody to Mycoplasma agassizii was determined by enzyme-linked immunosorbent assay (ELISA), and mollicutes species were detected in nasal flushes by polymerise chain reaction (PCR) Antibody prevalence varied among sites and years but was highest in 1998, exceeding 70% at two sites Only 11 tortoises (5%) were positive by PCR, and three species (M agassizii, testudineum, and a nonpathogenic Acholeplasma) were identified in nasal flush specimens. Nasal discharge, though rare (6% of tortoises), was significantly correlated with higher ELISA ratios, study site, and positive PCR status. Mortality events (n = 11) occurred on two of the three M agassizii-positive sites, no mortality was observed on the M agassizii-negative control site. However, none of the tested variables (ELISA result. study site, year, sex, presence of clinical signs, or carapace length) showed significant ability to predict the odds of death Mycoplasmal URTD is believed to be a chronic disease with high morbidity hut low mortality, and follow-up studies are needed to detect long-term effects.