The work described in this paper was designed to evaluate the relevance of in vitro skin penetration studies of peptides across rat skin. The apparent penetration of three peptides, enkephalin, elcatonin and insulin, in the presence of enhancers was not seen in the in vitro method using Franz diffusion cells. However, when a protease inhibitor was mixed in the receptor fluid, the penetration of enkephalin and insulin was observed. Although insulin penetrated in the presence of enhancers, the penetration was extremely small in quantity and the cumulative amount did not increase with time. When the degradation of peptides in the receptor fluid of Franz cell was estimated, these peptides, especially enkephalin and insulin, were rapidly hydrolyzed and were almost completely lost within 3 h in the absence of an inhibitor, while elcatonin was slowly degraded. The addition of protease inhibitors, such as gabexate (20 mM), camostat (20 mM) or bile salt (taurocholate and deoxycholate, 10 mM), to the receptor fluid inhibited the degradation to a considerable extent, with the first-order rate constants decreased to one-tenth compared with the contants without inhibitors. From the inhibitory study using specific inhibitors, it was clarified that enkephalin and elcatonin were mainly hydrolyzed by aminopeptidases, endopeptidases and serine proteases in the viable skin. Consequently, the results obtained from the in vitro penetration studies without inhibitors did not reflect reliable penetration data.Thus, effective protease inhibitor(s) should be used to obtain the data corresponding to the in vivo transdermal experiment. This methodology will provide a means to eliminate the confounding effect of metabolism in permeation experiments.