To unravel the molecular regulation of renal transcellular Ca²⁺ transport, a murine distal convoluted tubule (mpkDCT) cell line derived from distal convoluted tubules (DCT) microdissected from a SV-PK/Tag transgenic mouse was characterized. This cell line originated from DCT only, as mRNA encoding for the DCT marker thiazide-sensitive Na⁺/Cl^- cotransporter was expressed, while mRNA encoding for the connecting tubule and collecting duct marker aquaporin 2 was not detected, as determined by reverse transcriptase PCR. mpkDCT cells expressed mRNA encoding the Ca²⁺. channels TRPV5 and TRPV6 and other key-players necessary for transcellular Ca²⁺ transport, i.e. calbindin-D_9k, calbindin-D_28k, plasma membrane Ca²⁺-ATPase isoform 1b and Na⁺/Ca²⁺ exchanger 1. Primary cultures of DCT cells exhibited net transcellular Ca²⁺ transport of 0.4 ± 0.1 nmol.h^-1.cm^-2, while net transcellular Ca²⁺ transport across mpkDCT cells was significantly higher at 2.4 ± 0.4 nmol.h^-1.cm^-2. Transcellular Ca²⁺ transport across mpkDCT cells was completely inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated Ca²⁺ channel inhibitors felodipine and verapamil. Using patch-clamp analysis, the IC₅₀ of ruthenium red on Na⁺ currents was between the values measured for TRPV5- and TRPV6-expressing HEK293 cells, suggesting that TRPV5 and/or TRPV6 are possibly active in mpkDCT cells. Forskolin in combination with IBMX, 1,25(OH)₂D₃ and 1-deamino-8-D-arginine vasopressin (dDAVP) increased transcellular Ca²⁺ transport, whereas PMA and parathyroid hormone (PTH) had no significant effect. In conclusion, the murine mpkDCT cell line provides a unique cell model to study the molecular regulation of transcellular Ca²⁺ transport in the kidney in vitro.