The interactions of 1-dodecyl-azacycloheptan-2-one (Azone), a penetration enhancer, with mouse skin were analyzed by fluorescence microscopy, solid-state 13C-CP/MAS-NMR spectroscopy and Attenuated Total Reflectance Fourier-transform infrared (ATR-FT-IR) spectroscopy. Ferulic acid was employed as fluorescent probe to observe the delivery pathway in the stratum corneum (SC) after treatment with Azone. Results suggested that the interaction between Azone and the SC occurs in the lipid domains as well as the protein domains. FT-IR measurements show that treatment with Azone results in significant shifts toward larger wavenumbers at v(as)CH2 and v(s)CH2, indicating an increased gauche conformational isomer content of lipid CH2. Further, a decrease of (13C)T1 values and a shift of the SC protein amide-II band to a short wavenumber were found when the SC was pretreated with Azone. It is concluded that Azone can partially convert the SC protein from an alpha-helix conformation to a beta-sheet conformation and loosen the aggregating SC keratins at room temperature.