Membrane-bound carbonic anhydrase (CA) facilitates acidification in the kidney. Although most hydratase activity is considered due to CA IV, some in the basolateral membranes could be attributed to CA XII. Indeed, CA IV is glycosylphosphatidylinositol anchored, connoting apical polarization, but CA IV immunoreactivity has been detected on basolateral membranes of proximal tubules. Herein, we determined whether CA XII mRNA was expressed in acidifying segments of the rabbit nephron. The open reading frame of CA XII was sequenced from a rabbit kidney cortex cDNA library; it was 83% identical to human CA XII and coded for a 355-amino acid single-pass transmembrane protein. Northern blot analysis revealed an abundant 4.5-kb message in kidney cortex, medulla, and colon. By in situ hybridization, CA XII mRNA was expressed by proximal convoluted and straight tubules, cortical and medullary collecting ducts, and papillary epithelium. By RT-PCR, CA XII mRNA was abundantly expressed in cortical and medullary collecting ducts and thick ascending limb of Henle's loop; it was also expressed in proximal convoluted and straight tubules but not in glomeruli or S3 segments. FLAG-CA XII of ~40 kDa expressed in Escherichia coli showed hydratase activity that was inhibited by 0.1 mM acetazolamide. Unlike CA IV, expressed CA XII activity was inhibited by 1% SDS, suggesting insufficient disulfide linkages to stabilize the molecule. Western blotting of expressed CA XII with two anti-rabbit CA IV peptide antibodies showed no cross-reactivity. Our findings indicate that CA XII may contribute to the membrane CA activity of proximal tubules and collecting ducts.