This is the first direct potentiometric determination of intracellular bicarbonate concentration. The new method involves the use of a double-barrelled HCO3 −-selective liquid ion-exchange microelectrode that permits the simultaneous determination of intracellular [HCO3 −] and membrane PD of single cells. The mean in situ intracellular [HCO3 −] of single striated muscle fibers was 4.4±0.3 mM/l in the frog and 12.6±0.6 mM in the rat. Both values are inconsistent with a Donnan equilibrium distribution and can be accounted for by an active HCO3 − influx or an active H+ efflux. During progressive acute hypercapnia there is an accumulative build-up of intracellular bicarbonate in rat skeletal muscle. The increase in intracellular [HCO3 −] with hypercapnia is strictly proportional to the associated increase in plasma [HCO3 −], thus maintaining a constant ratio of extracellular: intracellular [HCO3 −]. Using the directly measured [HCO3 −] in cell water, we calculate a cell pH of 7.00 for frog fibers and of 7.14 for rat fibers, both values being about 1.1 pH units on the alkaline side of those predicted for a Donnan equilibrium distribution of H+ ions across the cell membrane.