We tested the hypothesis that cytosolic and membrane-associated carbonic anhydrase (CA IV) are involved in renal urinary acidification and bicarbonate reabsorption in rainbow trout. Using homology cloning techniques, an 1137 base pair cDNA was assembled that included an open reading frame encoding for a deduced protein of 297 amino acids. Phylogenetic analysis revealed that this protein was likely a CA IV isoform. Using this sequence and a previously described trout cytosolic isoform [tCAc (13)], tools were developed to quantify and localize mRNA and protein for the two CA isoforms. Unlike tCAc, which displayed a broad tissue distribution, trout CA IV mRNA (and to a lesser extent protein) was highly and preferentially expressed in the posterior kidney. The results of in situ hybridization, immunocytochemistry and standard histological procedures demonstrated that CA IV was likely confined to epithelial cells of the proximal tubule with the protein being expressed on both apical and basolateral membranes. The CA IV containing tubule cells were enriched with Na⁺/K⁺-ATPase. Similar results were obtained for tCAc except that it appeared to be present in both proximal and distal tubules. The levels of mRNA and protein for tCAc increased significantly during respiratory acidosis (hypercapnia). Although tCA IV mRNA was elevated after 24 h of hypercapnia, tCA IV protein levels were unaltered. By using F3500, a membrane-impermeant (yet filtered) inhibitor of CA, in concert with blood and urine analyses, we demonstrated that CA IV (and possibly other membrane-associated CA isoforms) plays a role in urinary acidification and renal bicarbonate reabsorption.