Abstract: The role of stratum corneum (SC) and its lipid constituents in skin permeability was assessed in vitro in diffusion system combined with the determination of partition coefficients (PC) and DSC technique. 5-Fu and 1, 8-CN were used as the hydrophilic drug and penetration enhancer, respectively. The results showed that the PCs of 5-Fu were the same in SC, delipid SC, full skin and SC-stripped full skin, but the diffusion coefficients (D) of 5-Fu were different from each other. They were 1.32±0. 37×10^-7, 13.71±5.12×10^-7, 1.01±0.31×10^-7 and 540. 85±50.99±10^-7 cm²/h, respectively. When the skin membranes were pretreated with 1,8-CN for 12 h, the PCs were reduced to similar extent. However, the D was increased significantly in SC and in full skin, there was a small increase for delipid SC. No change was found in the D of stripped skin. DSC analysis indicated that 1, 8-CN might change the thermal behaviour of SC lipid and the influence disappeared when SC was extracted with organic solvent. These results suggest that SC was the main barrier for 5-Fu diffusion through human skin, and the intercellar lipids played an important role for the hydrophilic drug penetration regardless of the presence of the enhancer in SC.