Mycoplasma iguanae was the suspected etiology of spinal disease in feral iguanas (Iguana iguana) from Florida. In an experimental infection study, juvenile iguanas were inoculated with M. iguanae intravenously or by instillation into the nares. Blood samples obtained at intervals postinoculation were all culture negative for mycoplasma. Gross anatomic and histologic findings at necropsy 12 wk postinoculation were unremarkable. Mycoplasmas were cultured in high numbers from the posterior choanae and upper trachea of some inoculated and control iguanas at necropsy. The 16S rDNA gene sequence of these isolates revealed they were all a previously undescribed strain, Mycoplasma insons proposed species nova. M. iguanae. Mycoplasma iguanae was not recovered from the conjunctivae, choanae, trachea, lung, coelomic cavity, blood, heart, liver, spleen, limb joints, brain, or spinal cord of inoculated iguanas, and the iguanas did not seroconvert. We conclude that M. iguanae is unlikely to be an agent of acute disease in iguanas and that M. insons can be considered as normal flora in the respiratory tract of iguanas.