High molecular weight inhibitors of carbonic anhydrase were synthesized and tested in isolated perfused rat kidneys. One such inhibitor, dextran-bound inhibitor (DBI), had a mean molecular mass of 6700 daltons. Its renal clearance was equal to the clearance of inulin and its intrarenal volume of distribution was close to that of inulin. The maximal effect of DBI on bicarbonate excretion was the same as that of acetazolamide. This action could not be attributed to breakdown products of DBI. DBI does not enter erythrocytes. Another inhibitor, extra-large inhibitor, had a mean molecular mass of 99,000 daltons. It was scarcely filterable. It did not increase bicarbonate excretion when added to perfusates in concentrations greater than effective concentrations of DBI. It is concluded that activity of carbonic anhydrase bound to luminal membranes of renal cells is critical for normal reabsorption of bicarbonate.