Opossum kidney (OK) cells (an epithelial cell line) were examined by flame photometry of cellular Na+ and K+ and by microfluorometric measurements of the intracellular pH (pHi) of single cells loaded with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). The work concentrates on defining resting pHi values under different experimental conditions and examines factors that contribute to the maintenance of resting pHi. To use nigericin to calibrate the intracellular response of BCECF, cellular K+ levels were measured by a null point analysis, and the stability and magnitude of cellular Na+ and K+ levels were determined vs. time. Resting pHi in medium without added CO2 was high when measured by null point analysis of the population (pHi 7.6) and from measurements of single cells that have recovered from an acid load caused by NH4 prepulse (pHi 7.76 +/- 0.03, n = 20 cells). In single-cell measurements, addition of CO2-HCO3- to the medium results in cellular acidification of the steady-state pHi by 0.35 +/- 0.04 pH units. In medium equilibrated with room air, the resting pHi is shown to be a dynamic steady state composed of net flux due to apical Na(+)-dependent transport (Na(+)H+ exchange) plus acidifying processes. It is concluded that although 5-[N-ethyl-N-isopropyl]amiloride (EIPA) inhibits the forward reaction of Na(+)-H+ exchange, EIPA is either ineffective as an inhibitor of the reverse reaction of Na(+)-H+ exchange or Na(+)-H+ exchange does not reverse measurably in the OK cells.