The appropriate use of acetazolamide in examining physiological function is based on the following: 1) the dissociation constant of the drug-carbonic anhydrase complex (10(-8) M); 2) the concentration needed to eliminate the catalyzed reaction (10(-4) M can be achieved readily in tissues by giving 20 mg/kg at no toxicity; and 4) the lack of known effects on any other systems at concentrations below 10(-3) M. Several problems have been identified and are analyzed concerning the use of acetazolamide and allied drugs. Concentrations greater than 10(-3) M have been used in vitro and occasionally in vivo, generating nonspecific effects. Experiments in which the substrate (CO2) is varied over a large range have been interpreted without regard to alteration in both the catalyzed and uncatalyzed rates, leading to conclusions that acetazolamide acts on a transport system other than carbonic anhydrase, while in reality the drug is acting on carbonic anhydrase at different levels of its catalytic rate. Since calculation of the uncatalyzed hydration of CO2 or dehydration of HCO3-in tissues involves some uncertainty, these rates are generally best defined by studying the dose-response curves of inhibitors and observing the residual activity after full inhibition. The kidney is an exception, since here the residual rate also involves an entirely separate process. Inhibitors other than acetazolamide are useful in ruling out nonspecific effects, and in gaining certainty of access to cells. Compounds closely akin to the inhibitors, but lacking action upon carbonic anhydrase, are also available as controls. It is emphasized that acetazolamide and other carbonic anhydrase inhibitors, when properly used, are highly specific probes of a single enzyme that has a wide variety of physiological functions.