BACKGROUND: Intestinal barrier dysfunction has been implicated in necrotizing enterocolitis (NEC), but has not been directly measured in human NEC. METHODS: Small intestines removed during surgery were immediately mounted in an Ussing chamber. mRNA expression of tight junction (TJ) proteins was measured with RT-PCR. RESULTS: Fifteen infants were included, 5 with NEC and 10 with other diagnoses. Average transepithelial resistance (TER) was 11.61±1.65Ω/cm in NEC specimens, 23.36±1.48Ω/cm at resection margin, and 46.48±5.65Ω/cm in controls. Average flux of permeability marker mannitol was 0.23±0.06μMol/cm per h in NEC, 0.04±0.01 μMol/cm per h at resection margin, and 0.017±0.004 μMol/cm per h in control tissue (p<0.05). RT-PCR analysis showed marked decrease in mRNA expression of a TJ protein occludin in NEC affected tissue (p<0.03 vs. control). Additionally, mRNA expression of myosin light chain kinase (MLCK), an important regulator of TJ permeability, was increased in NEC specimens. CONCLUSION: These studies show for the first time that NEC intestinal tissue have increased intestinal permeability, even at grossly healthy-appearing resection areas. The increase in intestinal permeability in NEC appeared to be related in part to a decrease in occludin and an increase in MLCK expression. LEVEL OF EVIDENCE: Level 2.