In the absence of vasopressin, medullary thick ascending limb cells express a K+-independent, furosemide-sensitive Na+-Cl− cotransporter that is inhibited by hypertonicity. The murine renal specific Na+-K+-2 Cl− cotransporter gene ( SLC12A1) gives rise to six alternatively spliced isoforms. Three feature a long COOH-terminal domain that encodes the butmetanide-sensitive Na+-K+-2 Cl−cotransporter (BSC1–9/NKCC2), and three with a short COOH-terminal domain, known as mBSC1-A4, B4, or F4 (19). Here we have determined the functional characteristics of mBSC1-A4, as expressed in Xenopus laevis oocytes. When incubated at normal oocyte osmolarity (∼200 mosmol/kgH2O), mBSC1–4-injected oocytes do not express significant Na+ uptake over H2O-injected controls, and immunohistochemical analysis shows that the majority of mBSC1–4 protein is in the oocyte cytoplasm and not at the plasma membrane. In contrast, when mBSC1–4 oocytes are exposed to hypotonicity (∼100 mosmol/kgH2O), a significant increase in Na+uptake but not in 86Rb+ uptake is observed. The increased Na+ uptake is Cl− dependent, furosemide sensitive, and cAMP sensitive but K+independent. Sodium uptake increases with decreasing osmolarity between 120 and 70 mosmol/kgH2O ( r = 0.95, P < 0.01). Immunohistochemical analysis shows that in hypotonic conditions mBSC1-A4 protein is expressed in the plasma membrane. These studies indicate that the mBSC1-A4 isoform of the SLC12A1 gene encodes a hypotonically activated, cAMP- and furosemide-sensitive Na+-Cl− cotransporter. Thus it is possible that alternative splicing of the BSC1 gene could provide the molecular mechanism enabling the Na+-Cl−-to-Na+-K+-2Cl−switching in thick ascending limb cells.