The purpose of the present studies was to examine the renal distribution and functional properties of Na(+)-HCO(3)(-) cotransporter type 4 (NBC4), the latest NBC isoform to be identified. Zonal distribution studies in rat kidney by Northern blot hybridization and RT-PCR demonstrated that NBC4 is highly abundant in the outer medulla and cortex but is low in the inner medulla. Nephron segment distribution studies indicated that NBC4 is predominantly expressed in the medullary and cortical thick ascending limb of the loop of Henle. Using specific primers on the basis of the published sequence (GenBank accession no. AF-207661), a full-length NBC4 variant was cloned from human liver and examined. The sequence of this variant (called NBC4e) is shorter by 86 amino acids vs. the published sequence. Xenopus laevis oocytes injected with the full-length NBC4e cRNA were compared with NBC1-expressing oocytes. Although exposure of NBC1-expressing oocytes to CO(2)/HCO(3)(-) resulted in immediate hyperpolarization, the NBC4-expressing oocytes did not show any alteration in membrane potential. NBC activity in oocytes, assayed as the Na(+)-dependent, HCO(3)(-)-mediated intracellular pH recovery from acidosis, indicated that NBC4 is a DIDS-inhibitable NBC. We propose that NBC4 is expressed in the thick ascending limb of the loop of Henle and mediates cellular HCO(3)(-) uptake in this segment.