The efflux transporter, P-glycoprotein the mucosal surface of intestinal enterocytes, is able to limit the absorption of a broad range of luminal xenobiotics including many drugs. Little is known about the physiological significance or natural substrates of intestinal PGP, although the observation that PGP-knockout mice develop a high incidence of inflammatory bowel disease in the presence of normal colonic microflora (1) suggests that it may protect the epithelial barrier from bacterial factors. To test this hypothesis, we investigated the effects of PGP activity on the response of epithelial cells to the gut commensal E.coli C25. Data are mean ± SEM. and significance tested by the Students t test. Exposure of the PGP-expressing colonic cell line, Caco-2 to 10⁸ colony forming units/ml of E.coli C25 for 4 hours had no significant effect on cell viability compared to untreated controls, as judged by the % of cells staining with trypan blue (1.9 ± 0.4% vs 3.1 ± 0.7%; n=3). Co-incubation with C25 and the PGP inhibitor GF120918 (5μM) (2), caused a 3.4 ± 0.4 fold (P ≤ 0.05, (n=3) increase in the level of necrosis compared to GF120918 controls. To further define the role of PGP in protecting epithelial cells from bacterial damage, the effects of C25 were compared in wild-type (MDCK-WT) and PGP-transfected (MDCK-PGP) kidney epithelial cells. C25 induced a significant increase in necrosis in MDCK-WT from 2.5 ± 0.5 to 11.2 ± 1.5 % (P ≤0.05); in contrast there was no effect on MDCK-PGP cells 1.5 ± 0.4% to 2.4 ± 0.5% (n = 8). Addition of GF120918 (5μM) to MDCK-PGP cells restored their sensitivity to C25 (10.1 ± 1.1 fold increase in necrosis compared to GF120918 controls). C25 also increased apoptosis, measured by DNA fragmentation ELISA, in MDCK-WT by 7.2 ± 1.0 fold versus control but had little effect in MDCK-PGP (1.4 ± 0.3 fold; n = 3). In preliminary studies, diluted culture fluid from E coli C25 inhibited net transport of digoxin across epithelial monolayers by 42.7% suggesting that this strain secretes factors that are transported by intestinal PGP. These data provide preliminary in vitro evidence that PGP is able to protect epithelial cells from the damaging effects of commensal bacteria. The mechanism by which PGP limits bacteria-induced apoptosis and cell death are currently unknown but may involve transport and exclusion of secreted bacterial factors. High levels of PGP expression in the lower gut may be important in maintaining homeostasis with the luminal microflora and may explain the development of inflammatory disease in this region following PGP deletion in mice.