Membrane-bound luminal carbonic anhydrase (CA) IV, by catalyzing the dehydration of carbonic acid into CO2 plus water, facilitates H+ secretion in the renal outer medullary collecting duct from the inner stripe (OMCDi). To examine the role of CA IV on H+ secretion, we measured net [Formula: see text] transport in perfused OMCDi segments and examined the effect on transport of two extracellular CA inhibitors, benzolamide and F-3500, aminobenzolamide coupled to a nontoxic polymer, polyoxyethylene bis(acetic acid) [synthesized and kindly provided by C. Conroy and T. Maren (C. W. Conroy, G. C. Wynns, and T. H. Maren. Bioorg. Chem. 24: 262–272, 1996)]. These agents would inhibit only the luminal CA enzyme. Dose titration curves for net[Formula: see text] flux were performed for each drug. Basal [Formula: see text] absorptive flux was 12 pmol ⋅ min−1 ⋅ mm−1in control segments and significantly increased to 16 pmol ⋅ min−1 ⋅ mm−1in segments from 3-day acid-treated animals. The concentrations of benzolamide and F-3500 that inhibited[Formula: see text] absorption by 50% were ∼0.1 and ∼5 μM, similar to the K i for CA IV inhibition by these agents (0.2 and 4.0 μM, respectively; T. Maren, C. W. Conroy, G. C. Wynns, and D. R. Godman. J. Pharmacol. Exp. Ther. 280: 98–104, 1997). Adding exogenous CA to the inhibitor in the perfusate nearly restored basal [Formula: see text] transport, suggesting that cytosolic CA II was not inhibited by these impermeant inhibitors. In OMCDi segments from acidotic rabbits, the concentrations of benzolamide and F-3500 that inhibited[Formula: see text] absorption by 50% were 50 and 500 μM, respectively, >100 times the K i for CA IV inhibition and for inhibition of [Formula: see text]transport in control tubules. Thus, in the OMCDi, doses of extracellular CA inhibitors that inhibited ∼50% of CA IV activity also comparably inhibited [Formula: see text] transport, indicating that H+ secretion depends in part on the availability of luminal CA IV activity. Acidosis substantially decreased the sensitivity of [Formula: see text]transport to CA inhibition.