The objective of this study was to examine the feasibility of using aerosolized fluorescent microspheres to examine particle distribution in the respiratory tract of birds following aerosol exposure. Adult domestic pigeons (Columbia livia domestica; n = 5 birds per microsphere size) were exposed to aerosolized monodispersed populations of various sized carboxylate microspheres (0.5, 1.0, 2.0, 3.0, 6.0, and 10.0 μm) for 30 min. For aerosol-exposure purposes, the birds were anesthetized with injectable anesthetics, intubated, and placed on positive-pressure ventilation using a mechanical ventilator. Immediately following aerosol exposure, the birds were euthanatized, and carcasses were preserved via intravenous infusion of modified paraformaldehyde/gluteraldehyde fixative (pH = 7.2 and 340 mOsm). Initial evaluation of microsphere distribution in air sacs (cranial and caudal thoracic and abdominal) and at the level of the ostia was performed using a stereoscopic microscope with an epifluorescent module. More detailed examination of the distribution of microspheres within the respiratory tract was achieved using a confocal scanning laser microscope with a krypton argon laser and a scanning electron microscope. The results from this study revealed that positive-pressure ventilation resulted in distribution of smaller sized fluorescent microspheres (sizes 1.0, 2.0, and 3.0 μm) throughout the pigeon's respiratory tracts, and these microspheres were in highest concentration in the secondary bronchi and ostia for all of the examined air sacs. The larger sized beads (6.0 and 10.0) were confined to the upper airway (trachea and primary bronchi). The results from this study allow for a better understanding of particle deposition following positive-pressure ventilation and aerosol exposure in birds.