The actual activity of ionised calcium (Ca2+) in gall bladder bile determined with an ion-selective electrode was significantly higher in patients with gall stone disease (n = 15) than in patients without gall stones (n = 10) (0.43 mmol/kg v 0.31 mmol/kg; p < 0.05). No change in the Ca2+ activity in any of the gall bladder bile samples was observed during equilibration with CO2. During titration with HCl/NaOH, however, the Ca2+ activity fell with increasing pH in a biphasic manner, with the breaking point occurring at a significantly lower median pH in patients with gall stones than in patients without (pH 7.1 v 8.2; p < 0.0001). The combination of a higher activity of calcium in bile and precipitation of bile salts taking place at a lower pH in patients with gall stone disease than in patients without gall stones suggests a major role for calcium and pH in the pathogenesis of gall stones. Strict anaerobic sampling is not necessary for the measurements of Ca2+ in gall bladder bile, because the Ca2+ was not significantly affected by the changes in pCO2. The metabolic studies suggest, however, that simultaneous measurements of the activity of Ca2+ and pH is important in order to interpret data for the calcium activity in gall bladder bile.