Carbonic anhydrase (CA) inhibitors lower the rate of aqueous humor (AH) secretion into the eye. Different CA isozymes might play different roles in the response. Here we have studied the effects of carbonic anhydrase inhibitors on cytoplasmic pH (pHi) regulation, using a dextran-bound CA inhibitor (DBI) to selectively inhibit membrane-associated CA in a cell line derived from rabbit NPE. pHi was measured using the fluorescent dye BCECF and the pHi responses to the cell permeable CA inhibitor acetazolamide (ACTZ) and DBI were compared. ACTZ markedly inhibited the rapid pHi changes elicited by bicarbonate/CO2 removal and readdition but DBI was ineffective in this respect, consistent with the inability of DBI to enter the cell and inhibit cytoplasmic CA isozymes. Added alone, ACTZ and DBI caused a similar reduction (0.2 pH units) of baseline pHi. We considered whether CA-IV might facilitate H+ extrusion via Na-H exchange. The Na-H exchanger inhibitor amiloride (1 mM) reduced pHi 0.52 +/- 0.10 pH units. In the presence of DBI, the magnitude of pHi reduction caused by amiloride was significantly (P < 0.05) reduced to 0.26 +/- 0.09 pH units. ACTZ similarly reduced the magnitude of the pHi reduction. DBI also reduced by approximately 40% the rate of pHi recovery in cells acidified by an ammonium chloride (20 mM) prepulse; a reduction in pHi recovery rate was also caused by ACTZ and amiloride. DBI failed to alter the pHi alkalinization response caused by elevating external potassium concentration, a response insensitive to amiloride but sensitive to ACTZ. These observations are consistent with a reduction in Na-H exchanger activity in the presence of DBI or ACTZ. We suggest that the CA-IV isozyme might catalyze rapid equilibration of H+ and HCO3- with CO2 in the unstirred layer outside the plasma membrane, preventing local accumulation of H+ which competes with sodium for the same external Na-H exchanger binding site. Inhibition of CA-IV could produce pHi changes that might alter the function of other ion transporters and channels in the NPE.