Pathogenicity of Gallibacterium anatis was studied in specific pathogen free layers in a controlled environment, applying the intranasal route for experimental infection. At 30 weeks, 37 hens were infected with 0.4 ml of 1.53 × 10(8) colony-forming units/ml suspension of G. anatis strain 07990 whereas equal numbers of hens were left uninfected for control. Following experimental infection, clinical signs and the number and weight of the eggs were recorded daily until 5 weeks post infection. Three birds from each group were killed at 3, 7, 10, 28 and 38 days post infection (d.p.i.) for necropsy and sampling for bacteriological and histopathological examinations. Additionally, necropsy examination was performed on all remaining birds at 38 d.p.i. G. anatis infection was found to have an immediate and severe effect on egg production, showing early and persistent colonization in respiratory and reproductive organs as well as in the gut of infected layers. In birds killed at various time points, G. anatis infection caused focal necrosis in the liver (1/37), folliculitis (2/37), pericarditis (3/37), haemorrhagic follicles (2/37), ruptured follicles (20/37), yolk in the body cavity (2/37) and egg peritonitis (1/37). The inflammation of the ovaries could be further confirmed by histopathological examination. Recovery of G. anatis from yolk at 10 d.p.i. indicates the potential of vertical transmission. Altogether, lesions reflect typical findings of G. anatis infection reported in natural cases. Thus, for the first time, lesions and the consecutive disease caused by G. anatis infection have been reproduced experimentally in a natural infection model.