We have spin labeled the stratum corneum (SC) with a lysine specific reagent, succinimidyl-2,2,5,5-tetramethyl-3-pirroline-1-oxyl-carboxylate spin label (SSL), to assess the dynamics and hydration degree of SC proteins by electron paramagnetic resonance (EPR) spectroscopy taking measurements directly from the intact tissue. Treating the SC with two percutaneous penetration enhancers, 8 M urea or 20% (v/v) 1-methyl-2-pyrrolidone (1 MP), destabilizes the proteins thus promoting more mobile and solvent-exposed protein conformations. Upon SC lipid depletion the nitroxide side chain becomes more solvent exposed, suggesting that the removal of hygroscopic substances in the extraction process favors more hydrated protein conformations. On the other hand, the treatments with 8 M urea or 40% (v/v) 1 MP did not alter significantly the fluidity in the SC lipid domain as assessed by the probe 5-doxyl stearic acid; these permeation enhancers, specially 1 MP, seem to increase the probe solubility in the solvent leading to a considerable fraction of spin label to be removed from the lipid domain.