Electrophysiological techniques were used in isolated perfused proximal convoluted tubules (PCT) to examine the effect of changes in bath protein concentration on paracellular properties and of active salt transport on relative sodium-to-chloride permeability (PNa/PCl). Control transepithelial potential difference (PD) and NaCl dilution potentials were determined in tubules perfused and bathed with an ultrafiltrate-like solution. PNa/PCl was calculated from the NaCl dilution potential. In the first series of experiments PD and PNa/PCl were redetermined in the same PCT following three experimental maneuvers known to inhibit active salt transport. Addition of 10(-5) M ouabain to the bath, removal of luminal glucose and alanine, and removal of luminal glucose, alanine, bicarbonate, and acetate reduced PD but did not alter the NaCl dilution potential, and therefore PNa/PCl was constant. Constant PNa/PCl in these experiments suggests a) that lowering bath NaCl concentration does not change transcellular current flow, b) that PNa/PCl reflects the ion selectivity of the paracellular pathway, and c) that the ion selectivity of the paracellular pathway is independent of active salt transport. In the second set of experiments PD and PNA/PCl were redetermined following addition of protein to the bath. Neither PD nor PNa/PCl was altered. Analyses of these data argue against modulation of paracellular permeability by bath protein in the in vitro rabbit PCT and suggest that the ion selectivity of the paracellular pathway is determined predominantly by junctional complexes rather than lateral intercellular spaces.