The genome of zebrafish D. rerio encodes two unlinked genes equally closely related to the SLC4A2/AE2 anion exchanger genes of mammals. One of these is the recently reported zebrafish Ae2 gene (Shmukler et al., Am J Physiol Renal Physiol 289:R835, 2005), now called Ae2.1. We now report the structural and functional characterization of the second zebrafish AE2 gene product, Ae2.2. The Ae2.2 gene of zebrafish LG24 encodes a polypeptide of 1232 aa in length, sharing 70% amino acid identity with zebrafish Ae2.1 and 67% identity with mouse AE2a. Zebrafish Ae2.2 expressed in Xenopus oocytes encodes a 135 kDa polypeptide that mediates bidirectional, DIDS-sensitive Cl(-)/Cl(-) exchange and Cl(-)/HCO3(-) exchange. Ae2.2-mediated Cl(-)/Cl(-) exchange is cation-independent, voltage-insensitive, and electroneutral. Acute regulation of anion exchange mediated by Ae2.2 includes activation by NH4(+) and independent inhibition by acidic intracellular pH (pHi) and by acidic extracellular pH (pHo). In situ hybridization reveals low-level expression of Ae2.2 mRNA in zebrafish embryo, most notably in posterior tectum, eye, pharynx, epidermal cells, and axial vascular structures, without notable expression in the Ae2.1-expressing pronephric duct. Knockdown of Ae2.2 mRNA, of Ae2.1 mRNA, or of both with nontoxic levels of N-morpholino-oligomers produced no grossly detectable morphological phenotype, and preserved normal structure of the head and the pronephric duct at 24 hpf. Key words: Chloride/bicarbonate exchanger, Xenopus oocyte, isotopic flux, in situ hybridization, N-morpholino oligomer.