Glicentin, the main component of enteroglucagon, has trophic effects on intestinal mucosa. It may also have an inhibitory effect on extraintestinal invasion of enteric bacteria. We have established an in vitro bioassay system for determining the effects of recombinant human glicentin on bacterial internalization by confluent enterocytes. An INT-407 cell line was serum-deprived for 2 days and was then treated on transwell filters for 24 h with a medium containing one of the following: glicentin 100 ng-1 microg/ml, glucagons-like peptide-2 (GLP-2) 1 microg/ml, 10% fetal bovine serum (FCS), or without any growth factors. Pure cultures of Salmonella enteritidis, Escherichia coli, and Enterococcus faecalis were introduced to the upper chambers of the filter units. Following 2 h of incubation the numbers of bacteria in the lower chambers were measured. Pretreatment of enterocytes with glicentin inhibited bacterial internalization compared to untreated or GLP-2 enterocytes. Glicentin was associated with inhibition of enterocyte internalization of enteric bacteria by a mechanism that might be related to the integrity of the enterocyte adhesive junctions and tight junctions and to the production of sIgA. Glicentin seems to have a function as a barrier-sustaining agent that inhibits extraintestinal invasion of enteric bacteria.