Bacterial lipopolysaccharide (LPS) has been speculated to facilitate bacterial translocation by a mechanism involving physical disruption of the gut mucosal barrier. Polarized, cultured intestinal epithelial cells (Caco-2 cells) were used to study the effect of LPS on enterocyte structure, viability, and susceptibility to bacterial invasion. Varying concentrations of biologically active LPS were incubated with enterocytes for 1 and 16 hr. LPS had no noticeable effect on enterocyte viability or morphology, as measured by uptake of vital dyes, by distribution of cytoskeletal filamentous actin, and by visualization of subcellular ultrastructure. Transepithelial electrical resistance was similar in enterocyte cultures incubated with LPS for 1 hr, but there was a noticeable decrease after 16 hr, indicating a loss of epithelial integrity after prolonged exposure to LPS. The effect of LPS on bacterial uptake was studied using six strains of enteric bacteria with varying abilities to invade Caco-2 cells: Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, Escherichia coli (2 strains), and Enterococcus faecalis. Electron microscopy showed enteric bacteria in intimate association with enterocyte apical microvilli, and internalized bacteria were consistently observed within cytoplasmic, membrane-bound vacuoles. Following a 1-hr incubation of individual strains of enteric bacteria with Caco-2 cells, numbers of viable intracellular bacteria varied significantly between individual bacterial strains, but numbers of intracellular bacteria were similar for each strain incubated with enterocytes exposed to 0, 10, and 100 micrograms LPS for 1 and 16 hr. Thus, although prolonged exposure to LPS might have some effect on enterocyte culture integrity (as measured by decreased electrical resistance), LPS had no discernible effect on enterocyte structure, viability, and susceptibility to bacterial invasion. These results suggested that LPS-induced bacterial translocation might not involve loss of epithelial viability, or facilitated entry of bacteria into intestinal epithelial cells.