Active absorption of calcium from the intestine and reabsorption of calcium from the kidney are major determinants of whole body calcium homeostasis. Two recently cloned proteins, CaT1 and ECaC, have been postulated to mediate apical calcium uptake by rat intestine and rabbit kidney, respectively. By screening a rat kidney cortex library with a CaT1 probe, we isolated a cDNA encoding a protein (CAT2) with 84.2 and 73.4% amino acid identities to ECaC and CaT1, respectively. Unlike ECaC, CaT2 is kidney-specific in the rat and was not detected in intestine, brain, adrenal gland, heart, skeletal muscle, liver, lung, spleen, thymus, and testis by Northern analysis or reverse transcription polymerase chain reaction. The expression pattern of CaT2 in kidney was similar to that of calbindin D(28K) and the sodium calcium exchanger 1, NCX1 by in situ hybridization of adjacent sections. Furthermore, the mRNAs for CaT2 and calbindin D(28K) were colocalized in the same cells. CaT2 mediated saturable calcium upraise with a Michaelis constant (K(m)) of 0.66 mM when expressed in Xenopus laevis oocytes. Under voltage clamp condition, CaT2 promoted inward currents in X. laevis oocytes upon external application of Ca2+·Sr2+ and Ba2+ but not Mg2+ also evoked inward currents in CaT2-expressing oocytes. Similar to the alkaline earth metal ions, application of Cd2+ elicited inward current in CaT2-expressing oocytes with a K(m) of 1.3 mM. Cd2+, however, also potently inhibited CaT2-mediated Ca2+ uptake with an IC50 of 5.4 μM. Ca2+ evoked currents were reduced at low pH and increased at high pH and were only slightly affected by the L-type voltage-dependent calcium channel antagonists, nifedipine, verapamil, diltiazem, and the agonist, Bay K 8644, even at relatively high concentrations. In conclusion, CaT2 may participate in calcium entry into the cells of the distal convoluted tubule and connecting segment of the nephron, where active reabsorption of calcium takes place via the transcellular route. The high sensitivity of CaT2 to Cd2+ also provides a potential explanation for Cd2+-induced hypercalciuria and resultant renal stone formation.