The effect of the penetration enhancers Azone, oleic acid, 1-dodecanol, dodecyl N,N-dimethylaminoacetate (DDAA), and dodecyl N,N-dimethylaminoisopropionate (DDAIP) on epithelial membrane lipids was examined using human buccal cell membranes as a model for epithelial lipid bilayer. Buccal epithelial cells (BEC) were labeled with l,6-diphenyl-l,3,5-hexatriene (DPH), l-(4-(trimethylammonio)phenyl)-6-phenyl-l,3,5-hexatriene (TMA-DPH), and 8-anilino-l-naphthalene sulphonic acid (ANS) fluorophores to characterize enhancer-induced changes in the hydrophobic core, in the superficial polar head region, and on the exterior surface, respectively, with fluorescence anisotropy and fluorescence lifetimes. All the enhancers studied were found to decrease the BEC membrane lipid packing order in a concentration-dependent and time-dependent manner in the deep bilayer region, as shown by a 37–66% decrease in anisotropy. Oleic acid was also found to disrupt membrane lipids strongly in the polar head region, causing at least a 34% decrease in anisotropy values. Azone and DDAA were shown to alter molecular movement on the surface of the bilayers (24 and 19% decrease in anisotropy, respectively). The results suggest that interaction with membrane lipid domains is an important, but not the only, mode of action for the penetration enhancers studied.