The overall pulmonary disposition of various fluorescent probes was viewed by confocal imaging following intratracheal delivery in the rat in vivo. The green fluorescent dyes, coumarin-6, a 350 Da lipophilic molecule; calcein, a 623 Da hydrophilic molecule; or FITC-albumin, a 65000 Da hydrophilic molecule; were insufflated as a dry powder or instilled as a solution in the lungs of rat in vivo. Immediately, 2 or 24 h following delivery, the lungs were colored with sulforhodamine and fixed by vascular perfusion. The lungs were then removed, grossly sliced and examined by confocal laser scanning fluorescence microscopy. Coumarin-6 diffused within minutes across the trachea, airways and alveolar tissue but was also retained for hours in type II alveolar epithelial cells. The diffusion of calcein across the tissue was fast as well, with no particular affinity for specific cells. FITC-albumin slowly permeated the tissue. It remained in the airspaces for hours and was intensively captured by alveolar macrophages. Compared to the powder, the solution bypassed dissolution and therefore shortened the lag time for diffusion and cellular capture. The technique allowed to obtain an overview of the fate of fluorescent probes locally in each region of the lungs and highlighted the strong dependence of the localization behavior on physico-chemical properties of molecules as well as a capture by particular cells of the pulmonary tissue.