The principal mechanisms controlling the molecular permeability through the skin are associated to the intercellular membranes of stratum corneum (SC), the outermost layer of mammalian skin. It is generally accepted that an increase in fluidity of these membranes leads to a reduction of the physical barrier exerted by SC with a consequent enhancement in permeation of different compounds. It is known that water diffusion in SC increases with the increase in the water content in SC, Using the spin labeling method we evaluate the effect of hydration on the fluidity of intercellular membranes at three depths of the alkyl chain, Increase in the water content in SC leads to a drastic increase in membrane fluidity especially in the region near the membrane/water interface; the effect decreases on going deeper inside the hydrophobic core. Analysis of electron paramagnetic resonance (EPR) parameters as a function of temperature showed that the rotational motion at depth of the 16th carbon atom of the chain experienced a phase transition at 45 and 60 degrees C, These phase transition temperatures were not altered by changes in the water content of SC. A phase transition between 28 and 48 degrees C was observed from the segmental motion in the region near the polar headgroup (up to 12th carbon in the chain) and was strongly dependent upon the hydration of SC. Our results give a better characterization of the fluidity of SC, the main parameter involved in the mechanisms that control the permeability of different compounds through skin.12371615