1. Cell membrane potentials were measured with intracellular 3 M-CKl microelectrodes in isolated, perfused segments of the straight portion of the rabbit proximal tubule. 2. Under in vitro conditions simulating the in vivo situation, the transepithelial potential difference was about 1.6 mV, lumen-positive, and the basolateral membrane potential was 61 mV, cell negative. 3. Isomolar single ion substitutions in the bath (K+ for Na+, isethionate for Cl-, N-methyl-D-glucamine (NMDG+) for Na+, and Cl- for HCO3-) resulted in significant basolateral membrane potential changes only when [K+] was increased and [HCO3-] was reduced; in both cases the basolateral membrane depolarized. Cl- and Na+ substitutions with large monovalent ions did not change basolateral membrane potential. 4. Transepithelial potential changes in substitution experiments suggest that, at the paracellular pathway, PK greater than PNa greater than PNMDG, and PCl greater than Pisethionate. 5. It is concluded that the basolateral membrane of these cells is mainly K+-conductive and that electrodiffusional PNa and PCl are undetectable by this technique. 6. Addition of 1 mM-Ba2+ to the bath reduced basolateral membrane electro-diffusional PK, as evidenced by depolarization and by a reduction of the magnitude of the change in membrane potential produced by increasing bath [K+]. 7. The depolarization produced by lowering bath [HCO3-] appears to result from a reduction of electrodiffusional PK, since it is blocked by Ba2+. There is no need to postulate a conductive pathway for HCO3- or a related species.