Skin surface proteolytic activity in the living animal was determined by a sensitive, non-invasive methodology developed in our laboratory. A non- leaky well was constructed on the shaved back of an anesthetized guinea pig. The well contained the reaction mixture including the substrate 125I-S- carboxymethylated insulin B-chain (ICMI). The proteolytic activity was shown to be time-dependent. The activity was strongly inhibited by pepstatin A, indicating the involvement of aspartic proteinase(s) such as cathepsin D and/or E. Pretreatment of the skin with propylene glycol blocked the proteolytic activity. The present study demonstrates the presence of proteolytic activity located on skin surface using a unique, non-invasive method for in situ proteinase determination in the living animal.