Thiazide diuretics are specific inhibitors of the Na, Cl-cotransporter in the distal convoluted tubule (DCT). In addition to producing diuresis and natriuresis, they have hypocalciuric effect. Recently, two apical calcium channels have been identified: TRPV5 and TRPV6, both are expressed in the DCT. We studied the effects of thiazides on mouse renal calcium handling and renal gene expression of TRPV5 and TRPV6, as well as calbindin-D28k and calbindin-D9k, both are calcium transport facilitators located in the DCT. Upregulation of renal TRPV5 was found 4 h after intraperitoneal injection of chlorothiazide (CTZ) at both 25 and 50 mg/kg, but not at 100 mg/kg. Chronic treatment with CTZ at 25 mg/kg twice daily for 3 days, with or without salt supplementation of 0.8% NaCl and 0.1% KCl as drinking water, caused hypocalciuria, but the gene expression patterns were different. Without salt supplementation, mice developed volume contraction and there were no changes in gene expression. When volume contraction was prevented by salt supplementation, there was significant increase in gene expression of TRPV5, calbindin-D28k, and calbindin-D9k. Salt supplementation alone also induced significant upregulation in TRPV5, TRPV6, and both calbindins. The upregulation of TRPV5 by CTZ and salt supplementation and salt alone was further confirmed with immunofluorescent staining studies. Our studies suggest that thiazides induce hypocalciuria through different mechanisms depending on the volume status. With volume contraction, increased calcium reabsorption in the proximal tubule plays the major role. Without volume contraction, hypocalciuria is probably achieved through increased calcium reabsorption in the DCT by the activation of transcellular calcium transport system, and upregulation of apical calcium channel TRPV5, calbindin-D28k and calbindin-D9k.