Prior to weaning, young rabbits take up increased amounts of macromolecules across the intestine and into the circulation. The increased macromolecular uptake may be due in part to decreased intestinal proteolysis in these animals. To assess the effect of intestinal proteolysis on macromolecular uptake, we initially tested the basal level of intestinal trypsin-like activity in newborn and 4-week-old weaned animals. The newborn rabbits had significantly less trypsin-like activity in their small intestinal rinse fluid compared to the 4-week-old animals. Subsequently, we tested the effect of the protease inhibitor, aprotinin, on small intestinal rinse fluid trypsin-like activity and the intestinal uptake of bovine serum albumin (BSA). Newborn and 2-week-old rabbits were force-fed with aprotinin or phosphate-buffered saline (PBS) followed after several hours by BSA. Animals pretreated with aprotinin had significantly decreased levels of small intestinal rinse fluid trypsin-like activity and increased concentrations of immunoreactive BSA (iBSA) in plasma compared to PBS-pre-treated control animals. 4-week-old weaned rabbits force-fed with equivalent amounts (on the basis of body weight) of aprotinin and BSA had significantly decreased trypsin-like activity in small intestinal rinse fluid compared to control animals. However, plasma iBSA was not significantly different from that of PBS-pretreated controls. These findings suggest that the inhibition of intestinal proteolytic activity is associated with increased macromolecular uptake in young rabbits prior to weaning. At weaning, suppression of proteolytic activity was not associated with increased macromolecular uptake detectable with the same assay procedures used successfully in the newborn animals. Thus, processes which act to limit macromolecular uptake in the older animals were not reversed by an inhibitor of proteolysis.