A special chamber was constructed with the goal of controlling the process of aerosol infection of chickens with Mycoplasma gallisepticum (MG). The virulent Australian MG field strain Ap3AS was used in each of three experiments. The response to infection of layer-strain pullets was measured serologically, by the incidence and severity of gross lesions in tracheas and air sacs, and by the relative numbers of MG isolated from tracheas and air sacs 2 wk after challenge. In two of the experiments tracheal sections were assessed microscopically. Exposure to a nebulized, undiluted broth culture of MG for 10, 20, or 30 min produced uniformly severe lesions and serological responses. By contrast, results were less severe and less consistent when doses of up to 10(8) color-changing units (CCU) were injected directly into the abdominal air sacs. Gross air sac lesions were consistently produced in almost all pullets by exposure to an infectious aerosol containing 10(2)-10(3) CCU/liter of air for 10 min and an air flow rate of 40 liters/min. This can be achieved by nebulizing a 10(-3) dilution of a fresh, early stationary phase culture of MG strain Ap3AS. However, under the conditions of these experiments, this dose did not produce significant gross or histologic lesions in the trachea.