Studies involving micropuncture and microperfusion of single nephrons require the handling of very small fluid samples. To facilitate handling, the tubule fluids are normally manipulated under paraffin oil. Calcium and, to a lesser extent, magnesium and phosphate concentrations may change significantly when placed in hydrated paraffin oil. Fluids of alkaline pH (about 7.4), either intrinsic or because of loss of CO2, may very quickly lose calcium in paraffin oil. Evidence suggests that this is due to a reversible binding of calcium and perhaps magnesium to the aqueous-oil interface that is dependent on the pH of the sample. It is recommended that tubule fluid samples be kept acidic to prevent calcium loss. A convenient method is by equilibrating the paraffin oil, prior to placement of aqueous samples, with a CO2 tension sufficient to lower the sample pH to below 7.0.