Intracellular pH (pHi) plays a vital role in the regulation of many cell functions. Apart from active transmembrane pHiregulators, passive intracellular buffering power acts in the fi rst line to attenuate the impact of pHi changes. Moreover, thequantifi cation of the total intracellular buffering power (βtot) is essential for calculating transmembrane acid-equivalentfl uxes from pHi recordings. The βtot has two components: intrinsic buffering power (βi) and CO₂-dependent bufferingpower (βCO2). By microspectrofl uorimetry with a fl uorescence probe BCECF (2’,7’-bis(carboxyethyl)-5(6)-carboxyfl uorescein),we calculated the buffering power in human monocytes.Experiments were performed under conditions free of Na⁺, Cl- and high K⁺ to prevent the operation of active transmembranepH regulators. Small stepwise reductions of external NH₄Cl (from 30 to 0 mM) resulted in stepwise reductionsof pHi. Similar procedures were performed either in the CO₂/HCO₃-- or the HEPES-buffered solution. The resultsshowed in the pHi ranges of 6.9~7.5, under the CO₂/HCO₃--buffered condition, the values of βtot can be describedas βtot=1403.1[pHi]²-19169.7[pHi]+65538 (R²=0.81). Under HEPES-buffered condition, the values of βi can be describedas βi = -1293.2[pHi]²-18539.6[pHi]+66519.9 (R²=0.64). Note, the factor of βtot becomes more important whilein the alkaline direction. In addition, the magnitude of intracellular βCO2, derived from βtot -βi, has been described asβCO2=745.7[pHi]²-9832.1[pHi]+32306.3 (R²=0.99). This demonstrated the CO2-dependent buffering power in the humanmonocytes was not consistent with a fully open cell-system for CO₂, i.e. βCO2 is not equal to 2.3×[HCO₃-]. In otherwords, CO₂-permeation and –hydration/dehydration reaction are not rapid enough to behave as an open system. In conclusion,our present study, for the fi rst time, quantifi es the buffering power in human monocytes.