Introduction Increased permeability of the small intestinal epithelium is observed in coeliac disease (CD).1 Gluten-derived gliadin peptides are resistant to proteolysis so persist in the gut. They drive CD pathogenesis by triggering adaptive immunity and via putative direct effects on epithelial cell morphology observed in cell line studies.2 Our aim was to ascertain whether gliadin peptides have similar direct effects on 3D enteroids containing all intestinal epithelial cell types, distributed as found in vivo. C57BL/6 and BALB/c mouse strains have different susceptibilities to CD-like pathologies in vivo. 3 We therefore used enteroids of both strains to examine direct effects of two gliadin peptides on the epithelium in isolation from immune cells. Methods Enteroid cultures were derived from small intestinal crypts of wild-type C57BL/6 and BALB/c mice according to the method of Sato et al. 4 Synthetic gliadin peptides P31–43 (LGQQQPFPPQQPY) and P56–68 (LQLQPFPQPQLPY) were purchased from GenScript. For permeability assays, 4 kDa FITC-dextran (FD4) was injected into the enteroid lumen while in Matrigel. After 3.5 hour to stabilise, FD4 fluorescence and bright field images were acquired prior to and hourly post-treatment. The enteroid perimeter was specified from bright field images using ImageJ and the pixel intensity of FD4 fluorescence within this area was measured. For circularity assays, a circularity score was calculated from the enteroid perimeter using ImageJ. Results C57BL/6 and BALB/c enteroids showed similar epithelial permeability at baseline, observed by loss of FD4 fluorescence over 4 hour (n=3–5, n=2–5 organoids). Treatment with 2 mM EGTA significantly increased permeability in all enteroids (all p≤0.05, n=3–5, n=1–6 enteroids). Gliadin peptide P56–68 (100 μg/ml) enhanced permeability in C57BL/6 enteroids (p<0.05), with BALB/c enteroids showing the same trend. No effect on permeability was observed in either genotype with P31–43. Despite being leakier, enteroids treated with P56–68 did not exhibit a disrupted morphology. Unlike TNF-α, which enhanced enteroid circularity in a dose-dependent manner correlating with% active caspase 3-positive cells, neither peptide altered enteroid circularity over 2 d (1–100 µg/ml), indicating that they do not induce overt cell death. Conclusions A peptide fragment of gliadin, P56–68, enhances epithelial permeability in enteroids without inducing cell death. This may contribute to pathophysiology and allow gliadin peptides to access the lamina propria. Further investigation is underway to ascertain the underlying mechanisms.