OBJECTIVE To investigate the mechanisms by which proteolytic enzymes, such as trypsin, chymotrypsin, papain, and bromelain, are able to cross the intestinal mucosal barrier after oral administration to man. METHODS Filter-grown Caco-2 cell monolayers were incubated with proteolytic enzymes and then the transepithelial electrical resistance (TEER) and the transport of the paracellular marker fluorescein were monitored. The effects of the enzymes on the cells were investigated by light microscopy and by biochemical assays. Transport of intact proteases across the cells was verified by monitoring the proteolytic activity and MALDI-TOF mass spectroscopic identification of undegraded trypsin. RESULTS Depending on time, concentration, and side of exposure to Caco-2 cell monolayers, all proteases decreased the TEER and increased the transport of fluorescein. Some morphological and metabolic changes were observed. The effects were reversible, but until 24 hours after removal of the proteases. Under the conditions of this in-vitro model, approximately 10% of the apically applied dose reached the basolateral compartment as biologically active, non-degraded molecules. CONCLUSIONS Proteolytic enzymes were found to exert considerable effects on the barrier function of Caco-2 monolayers, facilitating the transport of normally non-absorbable compounds. This suggests the also reported, but so far unexplained, systemic absorption of proteolytic enzymes after oral administration in vivo may occur by self-enhanced paracellular transport.