The influences of different mechanisms of penetration enhancers (such as menthol, azone, ethanol and nonivarnide) regarding the percutaneous absorption and skin irritation of ketoprofen formulations through rat skin were investigated by in vitro and in vivo study. The skin irritation degree at the end of the experiment (10 h) was deterinined by pathologic biopsy and colorimetry methods. In vitro, the menthol showed the most potent enhancing effect. Furthermore, the enhancement effect of a combination of menthol and nonivamide was higher than that of their individual use alone. In vivo the formulation containing 0.05% nonivantide, 5% menthol and 20% ethanol showed a higher penetration rate and an acceptable degree of skin irritation compared to a commercial product (Formax plus gel containing 3% ketoprofen), indicating that it could be used in the clinical situation.