An equation was formulated that enabled the calculation of the concentration of free Ca2+ in a reaction medium containing calcium, magnesium (or manganese), and up to five metal chelators. The concentrations of all ionic and chelating species present in the incubation mixture could also be estimated. The equation, a polynomial to the sixth power with respect to free Ca2+, is expressed in terms of known equilibrium (formation) constants and concentrations of reactants. The calculated values of free Ca*+ were compared with those determined experimentally, using a calcium ion-specific electrode, and the two were found to be in excellent agreement. To illustrate the usefulness of the technique, we examined the inhibition by calcium of NAD+-linked isocitrate dehydrogenase, in the presence of ethylene glycol bis(&aminoethyl ether) N,N'-tetraacetic acid (EGTA), ADP, ATP, isocitrate, citrate, and magnesium. The effect of manganese on the concentration of free Ca*+ in solutions of calcium and EGTA was also described. It was found that manganese, but not magnesium, displaced calcium from the Ca-EGTA chelate resulting in a much higher than expected concentration of free Ca2+. This raises questions about the inhibition of the isocitrate dehydrogenase by physiological concentrations of calcium previously reported.