Zinc inhibits cAMP-stimulated Cl- secretion in T84 epithelia and stimulation of NHE3 activity in Caco-2 epithelia (Hogue et al, 2009). Zinc may ameliorate secretory diarrhea and so may be included in improved oral rehydration solutions (Hogue et al, 2009). We now report an unexpected action of zinc on the paracellular pore. MDCKII epithelial layers possess a cation-selective paracellular pathway of high conductance and have been used to study the molecular basis of paracellular cation selectivity (Anderson and Van Italie, 2009). In addition MDCKII layers display only limited rheogenic ion transport. Application of 10mM ZnCl2 to either the apical or basal bathing solutions lead to a time dependent decrease in transepithelial conductance. At 1 hr control resistance 80±3.7 Ω.cm2 (SEM n=6) was increased to 1572±374 Ω.cm2 (n=5, p<0.01), apical and 368±58 Ω.cm2 (n=6, P<0.001) basal additions. The current/voltage relationship after Zn2+ changed from a linear relationship in controls to markedly non-linear, with decreased conductance from apical application being enhanced by basolateral negative potentials and vice-versa. This is consistent with voltage-dependent block of the paracellular pore. Since paracellular fluxes of charged solutes are sensitive to the transepithelial p.d., (Carr et al, 2006) we investigated the effect of a transepithelial driving force using a Na (apical solution) :N-methyl-d-glucamine (isosmotic replacement of basolateral NaCl) gradient to generate a transepithelial p.d. ( Ψa-b = 40.0 ± 1.3 (n=12) mV ( basal side positive)) and determined basal to apical 45Ca2+ fluxes (Jb-a). In control conditions, Ja-b increased significantly from 126 ± 7 to 208 ± 19 nmol.cm-2.hr-1 (n=6) p<0.005, whereas after application of 10mM zinc, Jb-a was reduced from 119 ± 4 to 46 ± 14 nmol.cm-2.hr-1 (n=6) p<0.005. Ψa-b measured with a Na:choline gradient was increased from 26.0 ± 3.1 to 33.9 ± 4.2 mV(n=8, p<0.01) by 10mM zinc indicating a concurrent increase in apparent cation selectivity by zinc. Immunocytochemistry of zinc-treated monolayers (apical 10mM application, methanol fixed after ~1hr) with confocal imaging indicated no change in cell morphology or localisation of occludin or Zo-1, but an increased cytoplasmic content of claudin-2 was noted often in a peri-nuclear location. We conclude that zinc may act to block the paracellular pore in cation-selective leaky epithelia.