To estimate the contribution of the paracellular shunt pathway to cation-selective permeability in the hamster medullary thick ascending limb of Henle’s loop, we observed the effect of protamine, a selective blocker of paracellular conductance, on salt-diffusion voltage (dV<sub>T</sub>) in the isolated nephron segment perfused in vitro. When 300 µg/ml protamine was added to the lumen, the lumen-positive dV<sub>T</sub> generated upon reduction of the lumen NaCl concentration was decreased from 5.1 ± 0.9 to 0.8 ± 0.8 mV and the calculated Na<sup>+</sup>/Cl<sup>–</sup> permeability ratio was decreased from 1.40 ± 0.14 to 0.86 ± 0.08. Although the effect of protamine persisted after removal of the agent from the lumen, addition of 30 U/ml heparin, which neutralizes protamine, returned the dV<sub>T</sub> toward the control level. This effect was almost the same when the orientation of the imposed NaCl gradient was reversed. Protamine exhibited a similar effect on dV<sub>T</sub> in the presence of ouabain added to the bath. Protamine was without effect from the bath. Protamine did not affect the basel V<sub>T</sub> perfused with the control solution. Increased V<sub>T</sub> by decreasing perfusion pressure was inhibited by adding protamine from the lumen. These observations suggest that the paracellular pathway contributes to the cation selectivity of the medullary thick ascending limb. The cation selectivity of the paracellular shunt pathway may mainly account for the changes in V<sub>T</sub> which are either dependent on the luminal flow rate or transmural NaCl concentration gradient, while it may not contribute to the basal level of V<sub>T</sub>.