A primary component of the intestinal mucous layer that functions as a barrier to luminal bacteria is mucin, a high-molecular-weight glucoprotein. In addition, the mucous layer also contains other important elements such as phospholipids (PLs), which may effect bacterial translocation (BTL). It has been reported that mucin inhibits Escherichia coli translocation; however, the effect of PLs on intestinal permeability is still controversial. We have recently reported that the concentration of mucous PLs is higher in neonatal as compared to adult rabbits. The functional significance of these biochemical differences on BTL remains to be determined. The aim of this study was to evaluate the effect of PL and mucin composition on BTL in a human enterocyte-cell culture model. Human enterocyte Caco-2 cells were seeded in 24-well tissue-culture plates and grown for 14 days to confluence. The monolayers were pretreated with phosphate buffered saline as control, 10 mg/ml or 20 mg/ml mucin, 0.5 mM or 1.0 mM PL mixture based on neonatal (NPL) and adult (APL) composition, and 10 mg/ml mucin with 0.5 mM either APL or NPL mixtures 30 min before a 120-min incubation period at 37 °C with 108 colony forming units (CFU) of E. coli C25. Non-internalized bacteria were killed by the addition of gentamicin. Internalized bacteria were quantified by counting CFU from enterocyte-cell lysates grown on MacConkey's agar. Mucin inhibited bacterial internalization, while both compositions of PLs promoted it. Mucin added to the PL solution also diminished the stimulatory effect of PLs on bacterial internalization. These results indicate that the increased concentration of PLs found in the intestinal mucous layer of neonates, and/or the alteration in the balance between PLs and mucin, may play a role in the increased BTL seen in neonates.