A system to simultaneously measure the total deposition of four different sizes of monodisperse microspheres in normal and damaged lungs of rats was developed and tested. The system reproducibly measured the deposition of microspheres in control rats, and the procedure was shown to be sufficiently sensitive to measure ozone‐induced changes in deposition rates. Rats exposed to 1.2 ppm ozone 6 h/d for 2 consecutive days showed greater deposition of the 1.09‐μm‐, 2.02‐μm‐, and 2.99‐μm‐ but not of the 0.48‐μm‐diameter microspheres when compared to controls. After 8 consecutive days of exposure to the same concentration of ozone, there were no differences in deposition rates between control and ozone‐exposed rats. Respiratory physiology and lung histopathology data provided evidence that subtle changes in the airway architecture and/or aerodynamics were likely to be responsible for the differential deposition rates as a function of the duration of ozone exposure.