To determine the effects of early antigen exposure on the maturation of the gastrointestinal mucosal barrier, rat pups were divided into three groups at the age of 14 d. In addition to normal maternal milk, group CM (n= 24) received daily a gavage feed of cow's milk and group PH (n= 20) a whey protein hydrolysate during the experimental feeding period (14–20 d). Controls (n= 15) remained on maternal milk only. At 21 d, when “gut closure” normally occurs, intestinal absorption of horseradish peroxidase (HRP), was examinedin vitroin Ussing chambers. The absorption of intact HRP [geometric mean (95% confidence interval)] was significantly higher in group CM [35.3 (16.7, 74.7) ng.h−1.cm−2] than in group PH [5.2 (1.4, 19.5) ng.h−1cm−2] and in controls [3.4 (0.8, 15.1) ng.h−1. cm−2;F= 5.54,p= 0.006]. The absorption of degraded HRP was comparable in all groups. There were no modifications in electrical parameters in association with increased mucosal permeability to HRP. Furthermore, in group CM electron-microscopic studies disclosed accumulation of HRP in the cytoplasm of the epithelial cells and in the intercellular spaces where cell junctions remained unaltered. These results indicate that early administration of antigens delays the process of gut closure. They further suggest that continuously enhanced endocytosis of macro-molecules is induced by an insult to the mucosa as part of the host response to these antigens, irrespective of the protection afforded by maternal milk. (Pediatr Res 32: 301–305, 1992)