Methazolamide was determined in plasma, whole blood, and urine by a GLC-mass spectrometric method. Temporal patterns of methazolamide concentrations in plasma and red blood cells were obtained following single- and multiple-dose oral administration of the drug. The nonlinearity in the binding of the drug to the red blood cell carbonic anhydrase was evident from a comparison of plasma and red blood cells concentrations. The drug was cleared slowly from the red blood cells. The binding constants to the two isoenzymes of carbonic anhydrase were determined from the plasma and red blood cell concentrations and were in agreement with those determined by previous measurements. The half-life of elimination was 7.5 hr. The urinary recovery of unchanged drug was approximately 25% of the administered dose.