In vitro permeation studies with biological membranes often involve long, aqueous maceration of the tissue. The present investigation examined the possible effects of hydration on barrier integrity of Swiss mouse skin; using water, methanol, ethanol, and butanol as permeants and a previously developed procedure involving multiple, sequential permeation runs on each piece of skin. The permeation rate of water increased almost linearly up to 30 hr of hydration and then tended to level off. Transport rates of methanol and ethanol increased asymptotically and then plateaued at approximately 15 hr. These results contrast with earlier findings on hairless mouse skin where the permeabilities of these three compounds were unaffected by aqueous immersion. The permeation rate of butanol also increased during the first 15 hr of hydration but gradually declined over the next 25 hr. This result again contrasts with the hairless mouse species in which butanol permeability doubled in 10 hr and then plateaued. The species differences in the hydration profiles appear related to the vastly dissimilar pellages and, in the Swiss mouse, may indicate greater involvement of the transfollicular pathway.