Turnover of the Na(+)-glucose cotransporter in the apical membrane of intestinal absorptive cells elicits alterations in tight-junction structure including the appearance of intrajunctional dilatations. Paralleling these structural responses, epithelial permeability to ions and nutrient-sized solutes increases. However, it is not known how these observed permeability changes specifically relate to the structural alterations elicited by glucose. Using a hemeconjugated peptide tracer (MP-11; mol wt, approximately 1900), the present study shows that the glucose-elicited tight-junction dilatations are specific anatomical sites of junctional permeation. This peptide tracer penetrates tight junctions selectively at sites of dilatations and is detected focally within the paracellular space. This same tracer does not penetrate junctions when glucose is not present. A heme-conjugated macromolecule (horseradish peroxidase; mol wt, approximately 40,000) is excluded by both glucose-exposed and glucose-unexposed tissues. The results of this study show a paracellular pathway for small peptides that is regulated during Na(+)-glucose-activated absorption. It is speculated that the paracellular pathway may contribute to the meal-related oligopeptide absorption that is known to occur and has previously been wholly attributed to the transcellular route.