Although the zebrafish has been used increasingly for the study of pronephric kidney development, studies of renal ion transporters and channels of the zebrafish remain few. We report the cDNA cloning and characterization of the AE2 anion exchanger ortholog from zebrafish kidney, slc4a2/ae2. The ae2 gene in Linkage Group 2 encodes a polypeptide of 1228 aa exhibiting 64% aa identity with mouse AE2a. The exon-intron boundaries of the zebrafish ae2 gene are nearly identical to those of the rodent and human genes. Whole mount in situ hybridization detects ae2 mRNA in prospective midbrain as early as the 5 somite stage, then later in the pronephric primordia and the forming pronephric duct, where it persists through 72 hrs post-fertilization (hpf). Zebrafish Ae2 expressed in Xenopus oocytes mediates Na(+)-independent, electroneutral (36)Cl(-)/Cl(-) exchange moderately sensitive to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), is inhibited by acidic intracellular pH and by acidic extracellular pH, but activated by (acidifying) ammonium and by hypertonicity. Zebrafish Ae2 also mediates Cl(-)/HCO3 (-) exchange in Xenopus oocytes, and accumulates in or near the plasma membrane in transfected HEK-293 cells. In 24-48 hpf zebrafish embryos, the predominant but not exclusive localization of Ae2 polypeptide is the apical membrane of pronephric duct epithelial cells. Thus, Ae2 resembles its mammalian orthologs in function, mechanism, and acute regulation, but differs in its preferentially apical expression in kidney. These results will inform tests of the role of Ae2 in zebrafish kidney development and function.