<b><i>Background/Aims:</i></b> Fine-tuning of renal calcium (Ca²⁺) reabsorption takes place in the late distal convoluted and connecting tubules (DCT2/CNT) of the kidney via transcellular Ca²⁺ transport. Here, Ca²⁺ enters the cell at the apical side via the epithelial Ca²⁺ channel transient receptor potential vanilloid 5 and is subsequently extruded at the basolateral side by the concerted actions of the plasma membrane Ca²⁺ ATPases and the Na⁺/Ca²⁺ exchanger 1 (NCX1). NCX1 is responsible for ∼70% of basolateral Ca²⁺ extrusion. The aim of this study was to determine the predominant NCX1 variant in the kidney and its role in Ca²⁺ transport. <b><i>Methods:</i></b> DCT2/CNT specific tubules were used to show the abundance of NCX1 specific isoforms. Renal NCX1 variants were cloned from mouse kidney tissue. Human Embryonic Kidney 293(T) cells were transiently transfected with NCX1.3, and Fura-2 measurements and ⁴⁵Ca²⁺ uptake assays were performed to determine several characteristics of NCX1.3 in the reverse mode. <b><i>Results:</i></b> NCX1.3 was demonstrated to be the predominant NCX1 variant in the DCT2/CNT, next to NCX1.2 and NCX1.7. NCX1.3 could be inhibited by SN-6, an NCX-specific inhibitor, whereas stimulation of the cAMP/PKA or PKC-mediated pathway did not affect Ca²⁺ influx as measured in the reverse mode. Lowering intracellular Ca²⁺ concentrations resulted in a decreased Ca²⁺ uptake. <b><i>Conclusion:</i></b> NCX1.3 is the predominant NCX variant in the DCT2/CNT tubules. Its function is dependent on intracellular Ca²⁺ concentrations.