The present study is a comparison of the influences of glycerol, propylene glycol (PG), isopropyl myristate (IPM) and a combination of PG and IPM (1/1; w/w) on human stratum corneum (SC) by means of differential scanning calorimetry (DSC) and wide and small angle X-ray-diffraction (WAXD and SAXD). The effects of glycerol and PG on SC structure can be attributed to their functional groups. In DSC transition temperatures of lipid fractions are decreased whereas SAXD long distances of lamellar phases reveal an additional interference due to an integration into hydrophilic regions of hexagonally packed lipids (PG) or orthorhombically packed lipids (glycerol). The increased repeat distance is attributed to the polar character of both molecules. However, with IPM the long distance remains unaffected. IPM is integrated into the lipophilic regions of SC lipid matrix as concluded from an increase of WAXD reflections of orthorhombical lipids and a decrease of WAXD reflections of hexagonal lipids. The combination of PG/IPM affects SC microstructure in a specific manner. DSC shows a decrease in transition temperatures of the lipid fractions, although not as much as expected from the single substances. Additionally, the combination of IPM/PG affects the short distances of orthorhombically and hexagonally packed lipids in WAXD measurements similar as PG alone, whereas the long distance seems to remain unaffected as in the case of IPM pretreatment. Adjuvants with penetration enhancing potential reveal different effects on SC lipid microstructure, which have to be kept in mind in terms of formulating systems for transdermal administration.