The enhancement effect of oleic acid on the in vivo percutaneous absorption of mannitol, 6-mercaptopurine, and butylparaben was evaluated in rats. A deconvolution method was applied to estimate skin permeation from urinary excretion profiles of drugs. Oleic acid markedly increased skin permeation of mannitol and 6-mercaptopurine, while it slightly increased the permeation of butylparaben at the low dose but decreased it at the high dose. To elucidate the mechanism of enhancement of skin permeation by oleic acid, the estimated in vivo penetration profiles were analyzed based on a two-layer skin diffusion model with polar and nonpolar routes in the stratum corneum. With respect to the polar route, oleic acid did not change the diffusion parameter but increased the partition parameter, suggesting that it increased hydration of the stratum corneum and enlarged the aqueous pore pathway. On the other hand, oleic acid increased both the diffusion and partition parameters of 6-mercaptopurine in the nonpolar route. The mechanism of action of oleic acid was compared to those determined in a previous in vitro study using guinea pig skin. It was revealed that the basic mechanism of oleic acid for enhancing drug permeation was comparable between the present and previous studies, and that the difference in the apparent enhancement effect of oleic acid was due to the in vitro-in vivo difference in the degree of hydration of the stratum corneum and the diffusion length of the dermis layer.