BACKGROUND Microbial endotoxins are normally present in the gut, usually without apparent harmful effects, whereas systemically administered endotoxin impairs the mucosal barrier function. Our aim was to investigate whether in vitro exposure to bacterial lipopolysaccharide (LPS) could affect the intestinal barrier properties of the rat small intestine. METHODS Small-intestinal segments from rats were mounted in Ussing diffusion chambers, and the mucosal to serosal permeation of the marker molecules bovine serum albumin (BSA) and 51Cr-ethylenediaminetetraacetic acid (EDTA) was measured after addition of LPS to the mucosal or serosal side. RESULTS Mucosal exposure to LPS (0.01, 0.05, 0.25 mg/ml) had no effects on the permeation of BSA and 51Cr-EDTA, whereas when added to the serosal side at 0.05 or 0.25 mg/ml, LPS increased the marker permeation. CONCLUSIONS Serosal LPS exposure in vitro increased the intestinal permeability to the different-sized markers, whereas mucosal LPS did not, indicating that the mechanisms leading to intestinal barrier impairment can be initiated in the intestinal wall itself.