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pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO₃– ions, FRTL-5 cells regulate intracellular pH (pH_i) by an Na⁺/H⁺ antiport but HCO₃–-dependent exchangers cannot operate. We have investigated pH_i regulation (by microfluorimetry and the pH sensitive dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxy-fluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in kREBS—Ringer buffer (pH 7·4) with or without HCO₃– ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na⁺/H⁺ antiporter and the stilbene derivative disodium 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) for HCO₃ –-dependent mechanisms), ionic substitution and by NH₄⁺/NH₃ (10mm) acid loading. Basal pH_i was lower in the presence (7·3±0·058, mean±S.D., n= 14) than in the absence (7·59±0·078, n=10) of HCO₃ ions. In HCO₃ –-free media, cells recovered from acid load by 0·34±0·04 pH units in the first 2 min and finally reached a pH_i of 7·35±0·06. This recovery was Na⁺-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO₃^–-containing media, cells recovered from an acid load at a similar rate, but reached 99 ± 10% (n = 9) of the baseline pH; this recovery was also dependent on Na⁺ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0·06±0·02 and 0·18±0·04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min. Removal of Na⁺ from HCO₃^–-containing media acidified the cells (pH=–0·82±0·05, n=10) within 40 min; this acidification was partially blocked by either amiloride or DIDS. Removal of Cl^– alkalinized the cells (pH=+0·51 ± 0·06, n=10) after 40 min, and this alkalinization was totally prevented by DIDS. Furthermore, in the absence of Na⁺ and presence of amiloride, alkalinization was still seen on the removal of Cl^–, albeit at a diminished rate (i.e. pH = +0·25±0·05, n=8) after 40 min. In conclusion, FRTL-5 cells maintain pH_i by two Na⁺-dependent exchangers, one sensitive to amiloride, the other to DIDS, and a Na⁺-independent, Cl^–/HCO₃– mechanism.