Aerosolized fluorescent microspheres were used to study particle deposition in site-specific regions of the lung with confocal laser scanning microscopy. A nebulizer was used to aerosolize microspheres followed by passage through a heated discharging column to reduce static charge and to remove water surrounding each microsphere. Precoating of microspheres with albumin helped to minimize displacement during vascular fixation of the lungs. Confocal laser microscopy facilitated visualization of microspheres throughout the bronchial tree, ducts, and alveoli of the lungs. The use of fluorescent microspheres and confocal laser imaging provided distinct advantages compared with other methods to study lung particle deposition due to (1) the generation of single microspheres of uniform size by nebulization, (2) easy detection of microspheres in large slabs of microdissected lung tissues, (3) excellent resolution of tissue surfaces and microspheres for an infinite number of orientations and planes of section, and (4) the ability to visualize microspheres below fluid lining layers and on surfaces that could not easily be done by other methods of microscopy.