Protamine is known to inhibit cation selectivity of the paracellular shunt pathway in several leaky nephron segments. Ion permeability of the superficial (SF) proximal straight tubules (PST) is selective to Cl<sup>–</sup>, whereas that of the juxtamedullary (JM) PST is selective to Na<sup>+</sup>. Protamine was used to estimate the contribution of the paracellular shunt pathway to ion selectivity in these segments. PSTs were isolated from the kidneys of Japanese white rabbit and microperfused in vitro. The ratio of Na<sup>+</sup> to Cl<sup>–</sup> permeability (P<sub>Na</sub>/P<sub>Cl</sub>) was estimated from the diffusion potential (dV<sub>T</sub>) generated by NaCl gradients. When 300 µg/ml protamine was added to the lumen, P<sub>Na</sub>/P<sub>Cl</sub> was decreased from 0.33 to 0.26 in SF-PST and from 1.80 to 1.29 in JM-PST, respectively, as the results of inhibition of cation selectivity. Addition of 30 U/ml heparin to the lumen, which neutralizes protamine, returned the ratios toward the control levels. Protamine exhibited similar effect on P<sub>Na</sub>/P<sub>Cl</sub> in the presence or the absence of ouabain in the bath, indicating that the observed voltage deflections were independent of the transcellular active transport process. Although P<sub>Na</sub>/P<sub>Cl</sub> was different between SF- and JM-PST, protamine inhibits Na<sup>+</sup> permeability in both segments. Preferential Cl<sup>–</sup> permeability in the SF-PST may be intrinsic to the paracellular route of this segment. The mechanism of this anion selectivity remains to be established.