Macroelectrodes designed to measure the extracellular free Mg²⁺ and Ca²⁺ concentrations ([Mg²⁺]_o, [Ca²⁺]_o) may be used to determine Mg²⁺ and Ca²⁺ binding to extracellular buffers. This is important, as buffer concentrations may change physiologically or experimentally. A simplified calibration method allowed [Mg²⁺]_o and [Ca²⁺]_o > 50 µmol/l to be accurately measured. The method was used to determine the apparent dissociation constant, K_app (± SD) of Mg²⁺ binding to aspartate (22°C, 101.7 ± 22.5 mmol/l, n = 8; 44°C, 45.2 ± 8.3 mmol/l, n = 6), citrate (high affinity, 0.33 ± 0.14 mmol/l, n = 4; low affinity, approximately 80 mmol/l), malate (15.9 ± 1.0 mmol/l, n = 7) and Ca²⁺ binding to malate (10.3 ± 1.1 mmol/l, n = 7). Calculated and measured K_app for Ca²⁺ binding to malate were only in agreement if the concept of ionic equivalent was used to adapt the tabulated constants to the experimental conditions. For Mg²⁺ binding to aspartate, malate and citrate there was no or only limited agreement with the calculated K_app. These findings emphasise the difficulties involved in calculating free concentrations in biological solutions. It is concluded that it is more accurate to measure K_app at the appropriate ionic strength and temperature.