||The stratum corneum layer of skin represents the rate-limiting barrier for transdermal delivery of polar compounds. During stratification, protein differentiation and increased crosslinking density render the stratum corneum corneocytes practically impermeable. Furthermore, lipid differentiation and protein degradation result in a complex, heterophasic intercellular lipid matrix. In order to identify the contributions of specific stratum corneum components to polar solute diffusional barriers and pathways, solvent-enhanced permeation and corresponding alterations of stratum corneum biophysical properties were ascertained concurrently. The contributions of the corneocytes were established by reducing protein crosslinking density with the disulfide reducing agent, dithiothreitol (DTT). DTT-enhanced mannitol and sucrose permeation ranged from an average 3- to 10-fold and exhibited significant donor-dependence. Increased permeation was associated with an average 100-fold increase in protein free thiols, subtle alterations of stratum corneum protein biophysical structure, and little or no alteration of lipid biophysical structure. Specific compositional alterations of the stratum corneum lipid matrix were induced by short chain alcohols and hexane. Mannitol permeation through human epidermis increased linearly with alcohol chain length for 75% (v/v) methanol, ethanol, and 2-propanol. The enhanced permeation correlated with the extraction of nonpolar, liquid-crystalline phase lipids, polar, gel phase lipids, and water-soluble protein degradation products. The contributions of liquid-crystalline and gel phase lipids to barrier function were discerned using hexane and aqueous alcohol treatments, respectively. Following hexane treatment, mannitol and sucrose permeation increased less than 10-fold. Subsequent alcohol treatments resulted in 20- to 40-fold increases in permeation and exhibited minimal chain length dependence. Initial hexane treatments extracted significant amounts of liquid-crystalline phase lipids. The subsequent solvent treatments removed water-soluble compounds and alcohol-soluble, gel phase lipids. Thus, alcohol-enhanced polar solute permeation was attributed primarily to extraction of gel phase lipids and protein degradation products, not liquid-crystalline phase lipid extraction. Therefore, polar solute diffusional pathways through the stratum corneum include the corneocytes as well as the lipid matrix. More important, the rate-limiting barrier, which resides within the heterophasic intercellular lipid matrix, results from the contributions of both gel phase lipids and water-soluble protein degradation products to lipid biophysical structure.