The nephron segment responsible for the acetazolamide-insensitive fraction of renal bicarbonate reabsorption has not been clearly delineated. This study compares superficial and deep nephron bicarbonate reabsorption before and after acetazolamide at two dose levels (20 and 50 mg/kg per h) in mutant Munich-Wistar rats employing both cortical and papillary micropuncture and microcalorimetry. Systemic acid-base balance and right whole kidney glomerular filtration rate were similar in all groups examined. The effects of the two doses of acetazolamide were indistinguishable and resulted in a significant increase in whole kidney bicarbonate excretion that compared favorably with the fraction delivered out of the left papillary tip. Acetazolamide inhibited superficial proximal bicarbonate reabsorption by 80.0%, whereas reabsorption up to the deep loop of Henle was decreased by only 52% (P less than 0.001). Bicarbonate reabsorption that was insensitive to acetazolamide occurred in the superficial and deep loop of Henle and between the distal tubule and base collecting duct. Because water reabsorption in these segments could serve to generate transepithelial bicarbonate concentration gradients favorable for reabsorption, we attempted to minimize water abstraction by combined administration of mannitol and acetazolamide. During this condition a significant increase in bicarbonate delivery up to the deep loop of Henle was noted (52 vs. 65%), whereas superficial nephron reabsorption was not altered. Furthermore, an outwardly directed bicarbonate concentration gradient from the deep loop of Henle to vasa recta was demonstrated during acetazolamide (delta tCO2 = 20.9 +/- 3.3 mM), but was abolished during combined mannitol and acetazolamide administration (delta tCO2 = 3.5 +/- 0.9 mM). It is concluded that carbonic anhydrase inhibition results in a disparate effect on nephron bicarbonate reabsorption when juxtamedullary and superficial nephron segments are compared. Our findings suggest that a mechanism for residual bicarbonate reabsorption during acetazolamide administration may be passive reabsorption driven by favorable transepithelial concentration gradients.