Bile salts are potent inhibitors of bovine carbonic anhydrase and human carbonic anhydrase I and human carbonic anhydrase II. To further characterize the binding of bile salts to carbonic anhydrase, rate constants for the CO2 hydration reaction in the presence of deoxycholate, cholate, glycocholate and taurocholate were determined using stop-flow experiments. Values for the Michaelis-Menton dissociation constant for bovine carbonic anhydrase, human carbonic anhydrase I and human carbonic anhydrase II were found to be 5.2, 9.2 and 13.2 mmol/L, respectively. The inhibition constant values for the various bile salts tested ranged from 0.1 to 1 mmol/L for bovine carbonic anhydrase, 1.6 to 2.4 mmol/L for human carbonic anhydrase I and 0.09 to 0.7 mmol/L for human carbonic anhydrase II. Our results suggest a mechanism of noncompetitive carbonic anhydrase inhibition for bile salts. Bile-salt binding to carbonic anhydrases as measured by scanning molecular sieve chromatography resulted in an increase in partition radius, molecular volume and surface area. The partition radius increased from 24 A to 28 A in the presence of 2.5 mmol/L sodium deoxycholate at critical micelle concentration. As determined by sedimentation equilibrium measurements, approximately 1 gm of carbonic anhydrase will bind 0.03 gm of deoxycholate, suggesting three to six binding sites for bile salt on the carbonic anhydrase molecule. The conformational changes and inhibition of carbonic anhydrases resulting from bile-salt binding may be important to the regulation of enzymatic activity in tissues along the enterohepatic circulation; by limiting bicarbonate availability this interaction may also contribute to the metabolic derangements seen in patients with cholestatic liver disease.