An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate the transport pathway of the peptides and proteins, salmon calcitonin (sCT), insulin (INS), recombinant hirudin (rHAV2), and recombinant human growth hormone (rhGH), in pulmonary epithelium. Human lung adenocarcinoma A549 cells formed continuous monolayers when grown on the polycarbonate filters of Transwell plates. The transport of the peptides and proteins having MW of 3400-22,000 Da was studied under different conditions. The results showed that the apparent permeability coefficients (Papp) of these macromolecules across A549 cell monolayers ranged from 2×10−6 to 5×10−6cm s−1 and exhibited a good inverse correlation with molecular weight. No concentration, direction, or temperature dependence was observed in the permeation of sCT, INS, and rHAV2. While the Papp of rhGH in the BA direction (2.25×10−6cm s−1) was less than that in the AB direction at both concentrations (3.20×10−6 and 3.29×10−6cm s−1). The Papp values of rhGH were concentration and temperature independent in the AB direction. These findings suggest that the hydrophilic peptides and proteins used in this study, sCT, INS, rHAV2, and rhGH, appear to cross the A549 cell monolayers via a paracellular pathway by a passive diffusion mechanism.