Free (ionic) calcium in human serum was measured with a commercially available ion specific electrode, together with a saturated KCl salt bridge, a saturated calomel reference electrode, and a direct reading electrometer. Effects of sodium, potassium, and magnesium on the electrode values for calcium were assessed and a rationale demonstrated for the use of calcium standards containing 140 mmol of NaCl, 5 mmol of KCl, and 0.55 mmol of MgCl2 per liter. Trypsin, triethanolamine, and heparin bind calcium; therefore, their use is to be avoided. Variability is greatly decreased if the serum is from a fasting subject and if the collection tube is completely filled, transported on ice, and handled anaerobically. The range of free calcium for 86 apparently healthy adults was 1.175-1.375 mmol/liter when measured at 25°C. The precision (coefficient of variation) of duplicates was 0.6%; the long term precision 2.0%.