An electron-capture GLC assay for acetazolamide in biological fluids was developed. Extraction efficiency was 69-104%. The minimum detectable amount of acetazolamide was 10 ng/sample. Concentrations of acetazolamide were determined by GLC in blood, plasma, plasma water, and saliva after oral administration of a single 250-mg dose to five volunteers. Erythrocyte levels were calculated from whole blood and plasma data. Concentration of free drug in the plasma was measured in samples of plasma water obtained by microultrafiltration. Peak plasma levels of 10-18 microng/ml were reached 1-3 hr after the dose. At least 1 hr later, erythrocyte levels reached peak concentrations of 13-29 microng/ml. Over 31 hr, plasma levels declined more rapidly than erythrocyte levels. Saliva concentrations averaged 1% of those in plasma and decreased at a rate equal to that of plasma. Saliva levels were proportional to, but not equal to, water concentration. Saliva to plasma ratios were consistent for any given individual and, therefore, offer a means of monitoring drug dosage without resorting to frequent blood sampling.