Exocrine cells like alveolar type II (ATII) cells are subject to extensive membrane traffic between exo- and endocytotic compartments. ATII cells produce surfactant, store it in vesicles called lamellar bodies (LBs) and release it into the alveolar space. Beside this exocytotic activity, ATII cells are also involved in endocytotic processes, allowing surfactant recycling and the recycling of integral membrane proteins to the LBs. Here we aimed at investigating the exchange of lipidic and proteinaceous components between LB's limiting membrane and the plasma membrane. We visualized the limiting LB membrane by overexpressing the lysosomal associated protein LAMP-3 fused to GFP (LAMP-3-GFP) and the plasma membrane by overexpression of a red fluorescent protein extended by a farnesylation motive (DsRed-Farn). Under control conditions, LAMP-3-GFP was homogenously distributed over the limiting LB membrane. However, a minor fraction of LBs exposed also DsRed-Farn at discrete domains at LB's surface. Upon ATII cell stimulation with secretagogues (ATP, phorbol-12-myristate-13- acetate, ionomycine) the area of DsRed-Farn domains at LB's surface increased 2 to 4 fold within 20 min of stimulation. This increase remained unaffected by PAO, a blocker of clathrin-dependent endocytosis, but was almost abolished by blockers of clathrin-independent endocytosis, like filipin and indomethacin, as well as by inhibitors of intra-cellular vesicular transport like bafilomycine A1, wortmannin and LY294002. We conclude that the stimulation of ATII cells with secretagogues resulted in the activation of clathrin-independent vesicular transport pathway of DsRed-Farn-labeled membrane compounds to the limiting LB membrane, where they were integrated and formed discrete domains, co-localizing with LAMP-3-GFP. [Supported by DFG, Project D1402, and European Union, Pulmonet]