The polymerase chain reaction (PCR) was used to detect Mycoplasma gallisepticum in samples collected from the environment of experimentally or naturally infected poultry. Culture was also used in the experimental infections. Of 160 samples of food, drinking water, feathers, droppings or dust collected during experimental infection, 103 were positive using a M. gallisepticum-specific PCR (MG-PCR) and 68 were positive using a PCR (mycoplasma-PCR) that detects all species of the genera Mycoplasma, Spiroplasma, Acholeplasma and Ureaplasma. Six of these samples were also positive by culture. In environmental samples collected on a depopulated M. gallisepticum-positive turkey farm, three and two out of a total of 12 were positive by mycoplasma-PCR and MG-PCR, respectively. These results indicate the disseminating capacity of this mycoplasma and the possible use of PCR methods for epidemiological analyses and control of farm decontamination before the introduction of new birds.