Skin has various types of transporters and is a biochemically active organ. These aspects of skin influence the distribution of chemicals in skin and their elimination from skin. The biochemical and histological variations of the skin must be taken into account when conducting transdermal penetration research. Here we used hairless mouse skin to investigate the percutaneous absorption of chemicals in vitro from the stratum corneum (SC) side to the viable skin (VS) side (forward direction) and from the VS side to the SC side (backward direction). We examined the effects of molecular weight, lipophilicity (Log K_o/w), electric charge, and the molecular structure of penetrants. The penetration flux of verapamil hydrochloride (VRP) for the backward direction was 3.2 times larger than that for the forward direction. The flux values of benzoic acid (BA) and para-hydroxybenzoic acid (pHBA) for the forward direction were 2.1 and 4.6 times larger than those for the backward direction, respectively. This directional difference was caused by the active transporter for VRP, the histological distribution of BA solubility, and the intermolecular hydrogen bonding between pHBA and skin tissue in the stripped skin. Across intact skin, in contrast, there was no difference in the skin penetration profile between the forward direction and backward directions.