Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA), similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented towards the plasma, such that it can catalyze the dehydration of plasma HCO₃^-. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO₃^- dehydration. Homology cloning techniques were used to assemble an 1127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For comparison, a second cDNA (1107 bp) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish CA IV was detected in gill tissue, specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity, and demonstrated a limited degree of co-localization with Na⁺,K⁺-ATPase. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO₂ excretion in dogfish by catalyzing the dehydration of plasma HCO₃^- ions.