The fetal epidermal barrier undergoes rapid development during late gestation despite conditions injurious to the skin postnatally, i.e. prolonged exposure to water (urine) and noxious substances such as pancreatic chymotrypsin. Nevertheless, at birth, term newborns have a superb epidermal barrier. Concomitant with formation of the stratum corneum in utero, vernix caseosa forms a natural multifunctional cream separating the skin surface from the amniotic fluid with possible unique barrier properties. Therefore, we investigated the effect of native vernix, synthetic vernix, and Desitin on penetration of chymotrypsin, a proteolytic enzyme present in both developing epidermis and meconium. Alpha-chymotrypsin penetration through test materials was conducted in vitro using a modified Franz diffusion cell. The presence of alpha-chymotrypsin in vernix and a possible inhibitory effect of vernix on alpha-chymotrypsin activity were investigated. Vernix films significantly impeded chymotrypsin penetration compared with controls during 24-h exposure experiments. Alpha-chymotryptic activity in vernix was undetectable, and vernix showed no endogenous inhibition of such activity. Both synthetic vernix and Desitin significantly impeded alpha-chymotrypsin penetration compared with controls during 9-h exposure experiments. With respect to the developing epidermal barrier, these results are consistent with the hypothesis that vernix films retain endogenous (epidermal) chymotrypsin while preventing exposure to exogenous (pancreatic) chymotrypsin.