ABSTRACT. Silva C.C., Brandão M.D.M., Nascimento E.R., Almeida J.F., Abreu D.L.C., Barreto M.L., Soares M.V., Machado L.S. & Pereira V.L.A. [Mycoplasma gallinarum in laying hens with respiratory disease.] Mycoplasma gallinarum em galinhas poedeiras com doença respiratória. Revista Brasileira de Medicina Veterinária, 36(4):347-350, 2014. Curso de Pós-Graduação de Higiene Veteriná- ria e Processamento Tecnológico de Produtos de Origem Animal, Faculdade de Veterinária, Universidade Federal Fluminense, Rua Vital Brasil Filho, 64, Vital Brazil, Niterói, RJ 25230-340, Brasil. E-mail: email@example.com Mycoplasma gallisepticum (MG), M. synoviae (MS) and M. meleagridis (MM) are recognized as pathogens of indisputable concern for the Poultry Industry. These species of mycoplasms are often related to apparent or subclinical infection, causing acute or chronic disease in hens, turkeys and other birds. M. gallinarum has been considered a commensal microrganism. This case report describes the diagnosis M. gallinarum in an outbreak of respiratory disease in laying hens. It was analyzed a total of 25 birds from four flocks in two farms from Rio de Janeiro state, Brazil. The hens showed a similar clinical picture, with respiratory signs, increased mortality and decreased egg production. Necropsies were performed in some birds after euthanasia by atlanto-occipital dislocation. Gross lesions of conjunctivitis, tracheitis, aerossacculitis, perihepatitis and peritonitis were observed. In two of the flocks, the hens had also cyanosis and hepatic lesions. Tracheal swabs were collected, for PCR and isolation of mycoplasmas. For PCR, DNA extraction was done by phenol/chloroform method and generic primers for Mycoplasma spp. and specific primers for MG, MS and M. gallinarum were used A total of 25 isolates positive for Mycoplasma spp. and negative for MG and MS were subjected to growth in liquid and solid Frey´s media and seven were typing by immunoperoxidase as M. gallinarum. These isolates were confirmed as M. gallinarum by PCR specific. The absence of MG and MS and the detection of M. gallinarum in hens studied, suggested that the clinical manifestations and the lesions observed, could be related to this agent. Consequently, further evaluate of this M. gallinarum strain pathogenicity is needed.