Characteristics of 22Na+ fluxes through Na+ channels in luminal-membrane vesicles isolated from either pars recta or pars convoluta of rabbit proximal tubule were studied. In NaCl-loaded vesicles from pars recta, transient accumulation of 22Na+ is observed, which is inhibited by amiloride. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using either these membrane vesicles loaded with different anions, or an outwardly directed K+ gradient with a K+ ionophore valinomycin. The vesicles containing the channel show a cation selectivity with the order Li+ greater than Na+ greater than K+. The amiloride-sensitive 22Na+ flux is dependent on intravesicular Ca2+. In NaCl-loaded vesicles from pars convoluta, no overshoot for 22Na+ uptake is observed. Furthermore, addition of amiloride to the incubation medium did not influence the uptake of 22Na+ in these vesicle preparations. It is concluded that Na+ channels are only present in pars recta of rabbit proximal tubule.