Na⁺- nutrient cotransport-dependent regulation of paracellular permeability has been demonstrated in rodent intestine and human intestinal epithelial cell lines. In cell lines this regulation is associated with phosphorylation of myosin II regulatory light chain (MLC). However, the subcellular localization of phosphorylated MLC during this regulation has not been studied and regulation of paracellular permeability and MLC phosphorylation has not been studied in isolated human intestine. To evaluate these events in human jejunum, isolated mucosa was mounted in Ussing chambers, characterized electrophysiologically, and then immunostained using anti-phosphorylated MLC and anti-total MLC antisera. MLC phosphorylation was assessed by calculating the ratio of anti-phosphorylated MLC signal to anti-total MLC signal within defined regions. Transmucosal resistance of mucosae without active Na⁺-glucose cotransport was 37 ± 3% greater than that of mucosae with active Na⁺-glucose cotransport within 15 min. Quantitative double-label immunofluorescence showed that the phosphorylated MLC-to-total MLC ratio increased by 45 ± 4% within the perijunctional actomyosin ring when Na⁺-glucose cotransport was active. Thus regulation of transmucosal resistance by Na⁺-glucose cotransport is accompanied by increased MLC phosphorylation within the perijunctional actomyosin ring. These data support the proposed critical role of the perijunctional cytoskeleton in physiological regulation of human small intestinal paracellular permeability.