Electrophysiologic and isotopic techniques were used to characterize the anion permeabilities of isolated perfused rabbit proximal tubules (S2 segments). Tubules were differentiated into chloride- and sodium-selective populations by electrophysiologic rather than by anatomic criteria. The tubules were studied under conditions that inhibited active sodium transport. The isotopic chloride permeability coefficient was 5.5 +/- 0.6 X 10(-5) cm/s (n = 19) for chloride-selective tubules and 3.2 +/- 0.6 X 10(-5) cm/s (n = 15) for sodium-selective tubules. The isethionate permeability coefficient was 1.1 +/- 0.2 X 10(-5) cm/s (n = 23) and did not vary with sodium or chloride selectivity. The variation of oxyanion permeability (bicarbonate, isethionate, and cyclamate) relative to chloride resulted from changes in chloride permeability, per se, rather than any change in the oxyanion permeability. A consistent relation between bicarbonate and isethionate permeability permitted the permeability of bicarbonate ion to be estimated at 1.3 X 10(-5) cm/s. The mechanism of ion selectivity appears to be different for the two populations of tubules. Chloride-selective tubules appear to hve relatively small pathways that do not contain fixed-charge sites. In contrast, sodium-selective tubules may have permeation pathways that contain fixed negative-charge sites.