The present study was undertaken to investigate the regulation of intracellular pH in human thyroid epithelial cells and to characterize the kinetics of the acid-extruding processes operating in the absence and presence of HCO₃^-, Na⁺ and Cl^-. A dynamic technique of dual excitation microfluorimetry and the pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein was employed. The intracellular pH was 7.01 ± 0.27 (n = 29) and 6.94 ± 0.25 (n = 54) in the absence and presence of HCO₃^- respectively. Both in the absence and presence of HCO₃^-, the recovery from intracellular acid loads was not only due to an Na⁺/H⁺ exchange, but also to an Na⁺-dependent HCO₃^-/Cl^- exchange. In alkaline conditions caused by NH₄Cl pulsing, an HCO₃^-/Cl^- exchange was also found. The cells in HCO₃^- responded with a wide range of maximal hydrogen efflux rates in experiments where cells were either pretreated with 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid or incubated with amiloride. The heterogeneity might be due to subpopulations of thyrocytes in different metabolic states or at different points in the cell cycle. It is concluded that recovery from intracellular acidification in human thyroid cells is due to both Na⁺/H⁺ exchange and Na⁺-dependent Cl^-/HCO₃^- exchange even in nominally HCO₃^--free conditions, and that recovery from intracellular alkalinization is due to a Cl^-/HCO₃^- exchange which needs to be characterized further.