There are a large number of intestinal disorders in which tight junction barrier defects are associated with changes in the expression and/or localization of tight junction proteins. In particular, claudin-2 has been shown to be upregulated in a number of disease states, including inflammatory bowel disease. Expression of claudin-2 has been demonstrated to increase paracellular Na+ permeability without increasing permeability to large molecules. This finding has contributed to the hypothesis that claudin-2 forms paracellular size and charge selective pore-like structures. However, available measures to study barrier function of epithelial monolayers cannot resolve function of individual pores. To overcome this limitation, patch clamp recordings were used to define claudin-2 function at a single channel level. Tight junction patch clamp recordings reveal single claudin-2 channel openings. Claudin-2 expression reduces global measures of transepithelial resistance and induces a corresponding increase in the frequency, but not size of claudin-2 opening events detected in patch clamp recordings. Opening events were not detectable when electrodes were sealed on apical cell membranes away from tight junctions. Ion substitution analyses demonstrate that claudin-2 openings are cation selective and permeable to methylamine+ (radius 1.9 Å), but have low permeability to tetramethylammonium+ (radius 2.8 Å). Claudin-2 channel openings are blocked by cysteine derivatization of I66, a residue within the first extracellular loop of claudin-2, known to be an important determinant of claudin-2 pore properties. Similarly application of lanthanum, a known claudin-2 blocker blocks all single channel openings. These data show that claudin-2 size and charge selectivity are explained by the average behavior of a population of claudin-2 channels present in the tight junction. Furthermore, these data suggest frequency and duration of tight junction channel openings as potential mechanism whereby tight junction barrier function may be regulated in health and disease.