The metabolism of betamethasone 17-valerate was estimated using an artificial living skin equivalent (LSE). Betamethasone 17-valerate, betamethasone 21-valerate and betamethasone were measured by a normal-phase high-performance liquid chromatographic (HPLC) method. Betamethasone 17-valerate was added to the culture medium with or without LSE homogenate. Degradation profiles (%) of betamethasone 17-valerate remaining in the culture medium with skin homogenate did not differ from those without homogenate. However, the conversion of betamethasone 21-valerate to betamethasone was accelerated by skin homogenate, indicating that LSE has a sufficient level of esterase.