The concentration of free protons, [H+], is an important modulator of most cellular functions; therefore the regulation of the intra- as well as the extracellular proton concentration is essential for all organisms. The mathematical description of the actual buffer capacity was developed by Koppel & Spiro (1914). The actual buffer capacity is defined as the amount of acid or base that must be added to change the pH of the cytosol. Conventionally, it is related to a change of one pH unit. Relating the buffer capacity to a change in pH might be mathematically correct, but causes a priori a pH dependence, because the same change in pH corresponds to different changes in the proton concentration depending on the initial pH. Here, we challenge this concept and present a concept, which is based on the change in proton concentration instead of the change in pH. To validate the newly derived buffer ratio as well as the buffer capacity, we performed [H+] measurements in Xenopus laevis oocytes with ion-selective microelectrodes. The cells were reversibly acidified by the addition and removal of the weak acid butyric acid, respectively and following ionophoretically injected H+ into the oocyte to change the baseline [H+]. Using the Henderson-Hasselbalch equation in the linear form, [H+] ∙ [A-] = Ka ∙ [HA], which describes the dissociation of an acid or a base, we end up with the following expression for the intrinsic buffer ratio: rhoi = (A ∙ Ka)∕([H+] + Ka)2 and for the CO2/HCO3– dependent buffer ratio: rhoCO2 = [HCO3-]∕[H+]. These equations were confirmed by experimental data.