We studied Na+-coupled acid-base transport at the basolateral membrane of single, isolated, perfused rabbit proximal tubules by monitoring the time course of intracellular pH (pHi). The latter was determined using a microspectrofluorometric apparatus to alternatively excite the pH-sensitive fluorescent dye 2',7'-bis-2-carboxyethyl-5(and -6)-carboxyfluorescein (BCECF) at 440 and 490 nm, while the fluorescence emission, was measured at 530 nm. All experiments were conducted in the nominal absence of HCO-3 S1, S2, and S3 segments from both superficial and juxtamedullary nephrons were examined individually. We found that removing Na+ from both the lumen and bath (i.e., basolateral solution) caused pHi to fall from 7.24 to 6.75 in the superficial S1 segment (SS1), from 7.14 to 6.67 in the SS2, and from 7.09 to 6.69 in the SS3. Similarly, in juxtamedullary nephrons (J), bilateral Na+ removal caused pHi to fall from 7.25 to 6.76 in the JS1, from 7.16 to 6.71 in the JS2, and from 7.10 to 6.75 in the JS3. In all six proximal tubule subtypes, returning Na+ to the bath caused pHi to recover (i.e., increase). 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 50 microM), an inhibitor of HCO-3 transport systems, blocked this Na+-dependent pHi recovery in all three superficial subtypes and the JS3 but had no effect in either the JS1 or JS2. On the other hand, 50 microM ethylisopropyl amiloride (EIPA), an inhibitor of Na-H exchange, blocked the Na+-dependent pHi recovery in the JS1 and JS2 but had no effect in the JS3 or any of the superficial subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)