Alveolar type II (ATII) cells produce surfactant and release it into the alveolar space via exocytosis of lamellar bodies (LBs). On the other hand, various forms of endocytosis take place, enabling the recycling of surfactant as well as of integral membrane proteins to the LB. Here we investigated the trafficking of protein and lipid components of plasma membrane between the plasma and limiting LB membrane by over-expressing lysosomal associated membrane protein 3 fused to green fluorescence protein (LAMP-3-GFP) and farnesylated DsRed (DsRed-Farn). LAMP-3-GFP was homogenously distributed over the entire limiting LB membrane, whereas DsRed-Farn predominantly accumulated at the plasma membrane. However, in a minor LB fraction, DsRed-Farn was also found in discrete domains at its limiting membrane. Upon stimulation of ATII cells with secretagogues, the area of DsRed-Farn domains on LB surfaces increased 2 to 4 fold within 20 minutes of stimulation. This increase remained unaffected by phenylarsine oxide, an inhibitor of clathrin-dependent endocytosis, but was almost abolished by filipin and indomethacin, blockers of clathrin-independent endocytosis. It was also blocked by bafilomycin A1, wortmannin and LY294002, inhibitors of intra-cellular vesicular transport. We conclude that secretagogues facilitate the transport of plasma membrane components to LBs via a clathrin-independent vesicular transport pathway.