Synthesis of citrulline from ornithine, NH4+, and HCO3- by isolated pig liver mitochondria is inhibited by acetazolamide, a specific inhibitor of carbonic anhydrase, at the same concentrations which inhibit the mitochondrial matrix carbonic anhydrase. At an acetazolamide concentration sufficient to give complete inhibition of matrix carbonic anhydrase, the rate of citrulline synthesis is reduced by 71%, but no further decrease in citrulline is observed at higher concentrations of acetazolamide. Stimulation of O2 uptake by ornithine under conditions of maximal citrulline synthesis is also inhibited by acetazolamide. At pH 6.7, the ratio of citrulline synthesis is depressed relative to the rates observed over the range 7.2-7.7, and acetazolamide inhibits this rate by only 20%. These results support the hypothesis that the physiological role of carbonic anhydrase in liver mitochondria is to supply HCO3- as the substrate for the enzyme carbamoyl phosphate synthetase I, which provides the intermediate carbamoylphosphate in the rate-limiting step of citrulline synthesis. Since the uncatalyzed rate of CO2 hydration is rapid enough that it should not be rate-limiting for the carbamoylphosphate synthetase reaction, carbonic anhydrase appears to regulate access of HCO3- in the synthetase and so should be considered as one of the enzymes participating in the biosynthetic pathway leading to urea formation in the hepatocyte.