This study explored the effect of temperature on the diffusion and partition of nitroglycerin in the hairless mouse skin in vitro. The diffusion coefficient of the drug in the stratum corneum and viable skin agreed with the Arrhenius relationship with the activation energy of 69.1 (kJ/mol) and 46.1 (kJ/mol), respectively. The partition coefficient remained almost constant within the temperature range varied (12∼42 °C). The step change in the eenvironmental temperature from +10 °C to -20 °C range required about 0.6 hours before the skin permeation reached a steady state.