Pulmonary administration of drugs has recently gained attention because it exhibits numerous advantages compared to oral or intravenous administration. The administration of aerosols for inhalation to animals, however, remains a critical challenge and only a few methods of administration have been developed. Herein, we compare the regional distribution of aerosols in the lungs of wild type rats after pulmonary administration using three different methods: (a) The Penn-Century MicroSprayer® Aerosolizer; (b) an in-house designed aerosol generation system; and (c) the Aeroneb® Lab Micropump Nebulizer. Both the regional distribution and the fraction of aerosol deposited in the lungs were determined by means of radiolabelling of the aerosol followed by in vivo and ex vivo Positron Emission Tomography (PET) imaging and dissection and gamma counting. Endotracheal insufflation using the PennCentury MicroSprayer resulted in >85% of the administered dose accumulated in the lungs, with a non-uniform distribution of the radioactivity in different lobes and a low animal-to-animal reproducibility. Administration using the in-house designed and the Aeroneb nebulizers resulted in a uniform distribution over the lungs, but only a small fraction of the nebulized activity (ca. 0.1%) was deposited in the lungs.