We report on a disease in 27 birds (1 bird belonging to the order Coraciiformes, 3 to Piciformes, 4 to Galliformes, 7 to Psittaciformes, and 12 to Passeriformes) caused by fastidious mycobacteria. All birds were caged at the Antwerp Zoo and died suddenly between 1983 and 1994. Seventeen birds had no previous signs of disease, and 10 birds showed emaciation. Gross necropsy findings were generally nonspecific, but all the birds were smear positive for acid-fast bacilli (AFB). Histopathologic evaluation performed on 14 birds revealed predominantly intracellular AFB. Extracellular AFB were more abundant in advanced lesions, especially in necrotic areas. In the intestine the mucosal area was generally heavily infiltrated, suggesting an intestinal origin of the infection. There was extensive invasion of the lungs in most birds. In 11 birds sparse growth was obtained after at least 6 months of incubation on Lowenstein-Jensen medium or on Ogawa medium supplemented with mycobactin. Subculture was unsuccessful in all instances. The 16S rRNA gene sequence of the cultured organisms or tissues from seven birds revealed the characteristic signature sequence for Mycobacterium genavense. Direct bird-to-bird transmission in the zoo was unlikely, and the pathogenicity of M. genavense in birds seems to be limited. The source of M. genavense in nature and the epidemiology of the disease in birds remain obscure. As suspected for human cases of M. genavense infection, an oral route of infection has been suggested, and contaminated local water distribution systems may have been the source of the infection. Our study confirms that infections caused by M. genavense should be suspected in birds (especially in Passeriformes and Psittaciformes orders) that die suddenly without previous symptoms and that have AFB in tissues that are difficult to grow on conventional media.