An epidemic of pneumonia with fibrinous polyserositis and multifocal arthritis emerged in captive American alligators (Alligator mississippiensis) in Florida, United States, in 1995. Mycoplasma alligatoris sp. nov. was cultured from multiple organs, peripheral blood, synovial fluid, and cerebrospinal fluid of affected alligators. In a subsequent experimental inoculation study, the Henle-Koch-Evans postulates were fulfilled for M. alligatoris as the etiological agent of fatal mycoplasmosis of alligators. That finding was remarkable because mycoplasmal disease is rarely fatal in animals. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies produced by alligators in response to M. alligatoris exposure was developed by using plasma obtained from naturally infected alligators during the original epidemic. The assay was validated by using plasma obtained during an experimental dose-response study and applied to analyze plasma obtained from captive and wild crocodilian species. The ELISA reliably detected alligator seroconversion (P < 0.05) beginning 6 weeks after inoculation. The ELISA also detected seroconversion (P < 0.05) in the relatively closely related broad-nosed caiman Caiman latirostris and the relatively distantly related Siamese crocodile Crocodylus siamensis following experimental inoculation with M. alligatoris. The ELISA may be used to monitor exposure to the lethal pathogen M. alligatoris among captive, repatriated, and wild crocodilian species.An epidemic of mycoplasmosis emerged in a collection of American alligators (Alligator mississippiensis) in Florida in 1995 (4, 8). Thirty-three 200-to 300-kg adult male alligators died, and 13 moribund alligators were euthanatized within 1 month of the index case. Lesions observed at necropsy ranged from mild interstitial and peribronchiolar pneumonia to fibronecrotic pneumonia. Extrapulmonary complications included pericarditis, myocarditis, and multifocal arthritis. Mycoplasma alligatoris proposed sp. nov. was isolated from multiple tissues, blood, synovial fluid, and cerebrospinal fluid of affected alligators. In a pilot experimental inoculation study (5, 6), healthy alligators were inoculated with M. alligatoris strain A21JP2 T (ATCC 700619) to reproduce the disease and fulfill the HenleKoch-Evans postulates (11) for M. alligatoris as the etiological agent of synovitis, polyserositis, and pneumonia of alligators. The results were remarkable because, except for bovine and caprine pleuropneumonia, mycoplasmal disease is rarely fatal in animals.The origin of the 1995 epidemic remains unknown, but the disease may emerge under conditions of captivity. Crocodilians are frequently sold or traded among collections, exhibits, zoos, and ranches. Many collections include multiple species of crocodilians. Some crocodilians from commercial ranches are repatriated after "head-starting" hatchlings from eggs collected in the wild, a potential vector for catastrophic infection of wild populations if lethal disease emerges in captivity. A validated ser...