Epidermal enzymes play an important role in the process of differentiation of keratinocytes. The present preliminary in vitro study was undertaken to observe if topical enzyme treatment influenced permeation of compounds across the skin. Due to the noted function and importance of phosphatidylcholine metabolism during maturation of the barrier lipids, the effects of topical application of the phosphatidylcholine dependent phospholipase C enzyme (not present in epidermis) on skin penetration of three model drugs, viz. benzoic acid, mannitol and testosterone, were studied. Similar studies were also carried out using epidermal enzymes like triacylglycerol hydrolase, acid phosphatase, and phospholipase A2 (present in epidermis). Pretreatment of skin with phospholipase C significantly enhanced permeation of benzoic acid, mannitol, and testosterone relative to untreated skin. Triacylglycerol hydrolase (neutral) increased the penetration of mannitol 3-fold and had no effect on benzoic acid penetration. Topical application of acid phosphatase did not alter the permeation of any of these compounds. Phospholipase A2 significantly enhanced permeation of benzoic acid and mannitol while it did not have any effect on the penetration of testosterone. These results for the first time demonstrate that enzymes may remarkably affect and/or regulate the permeation of topically applied compounds.