The purpose of this study was to investigate intracytoplasmic pH (pHi) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pHi was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pHi values, determined in bicarbonate-free medium (extracellular pH: 7.40), were 7.25 +/- 0.02 (n = 23) and 7.17 +/- 0.04 (n = 30) for cultured PCT and DCTb respectively. After the acid-loading procedure, cultured proximal cells recovered their pHi by means of the classic Na+/H+ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pHi recovery was mediated by a Na+/H+ exchange present in the basolateral side. Moreover, at physiological initial pHi values, chloride removal from the apical solution caused the pHi to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pHi recovery was induced in sodium-free media by 15 mM HCO(-3) in the presence of an outward chloride gradient. This pHi change was completely abolished by 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na(+)-independent Cl-/HCO(-3) exchanger.