Marek's disease virus (MDV), which is the causative agent of Marek's disease (MD), is shed by infected chickens and transmitted to other chickens through the respiratory route. Experimental reproduction of MD has been commonly done either by intra-abdominal inoculation of cell-associated MDV or by exposure to MDV-infected 'seeder' chickens. The former method does not mimic the natural route of MDV infection, whereas the latter method suffers from lack of uniformity in the timing and amount of virus transmission from seeder chickens to susceptible birds. The aim of the present study was to establish an infection model of MDV that mimics the natural route of infection. Here we report that when chickens were exposed for 20 min to aerosols (particle size 1.91 microm) of cell-free MDV suspensions containing 1280 plaque-forming units/ml, which were generated using a nebulizer, pathological and clinical signs of MD were observed in 95%-100% of the aerosol-exposed chickens by 21 days post-infection (dpi). Chickens that were exposed to aerosols and sampled at 1, 2, 3, 10, and 21 dpi showed MDV replication as early as 1 dpi in lungs as well as in other tissues such as spleen and bursa of Fabricius. This infection model will facilitate the studies directed to elucidate MDV-host interaction at the site of virus entry.