In isolated perfused rat liver, urea synthesis is rapid and reversibly inhibited not only by the well-known carbonic anhydrase inhibitors acetazolamide, methazolamide and ethoxzolamide, but also by diuretics, like xipamide, mefruside, chlortalidone, and chlorothiazide. Furosemide was without effect. Similar to findings with isolated perfused rat liver, acetazolamide inhibits urea synthesis from ammonium ions in normal and cirrhotic human liver slices. Inhibition of urea synthesis by xipamide and acetazolamide is accompanied by a 70% decrease of the cellular citrulline content and the tissue levels of 2-oxoglutarate and citrate, suggesting a block of urea synthesis at a step prior to citrulline formation. At a constant extracellular pH (7.4), inhibition of urea synthesis by xipamide, mefruside and acetazolamide was overcome by increasing the extracellular concentrations of HCO3- and CO2 to above twice the normal values. This shows that inhibition of urea synthesis by these diuretics is not due to an unspecific inhibition of one of the urea cycle enzymes but is due to an inhibition of mitochondrial carbonic anhydrase and therefore due to an impaired HCO3- provision for mitochondrial carbamoylphosphate synthesis. It is concluded that the activity of mitochondrial carbonic anhydrase is required for urea synthesis also in human liver and that several diuretics impair urea synthesis by inhibition of mitochondrial carbonic anhydrase. The pathophysiological significance of these data is discussed with respect to the development of diuretics-induced hyperammonemia and alkalosis in liver disease.