This work was conducted on rats in two premises located in Niterói and Rio de Janeiro, Brazil. One is classified as conventional controlled and the other, conventional. The objective of the present study was to detect the presence of Mycoplasma pulmonis in animals with symptoms of respiratory disease and low reproductive performance. In the conventional controlled premises, 16 rats of Wistar-Furth strain were necropsied while in the conventional premises necropsy was performed on 12 rats of Hooded Lister strain. The clinical samples of lungs, trachea, oropharynx, middle ear, uterus and ovaries were subjected to culturing while the sera were tested for antibody detection. From 28 rats, 57.14% (16/28) were culture positive for M. pulmonis, being 81.25% (13/16) from the conventional controlled premises, and 25.00% (3/12) from the conventional premises. The ELISA test was carried out in 20 animals of both colonies. In the conventional controlled premises, 92.86% (13/14) were positive for M. pulmonis, and 7.14% (1/14) were suspicious, while in the conventional premises, 100% (6/6) of the samples were positive. The results confirmed that M. pulmonis was the etiologic agent of the disease that affected the rats under study, and that the ELISA positivity rated higher than culture.