The renal late distal convoluted tubules and connecting tubules are sites for the fine-regulation of Na⁺- and Ca²⁺-reabsorption. The role of these segments in Na⁺- and K⁺-homeostasis is possibly underestimated, as the tubules are technically difficult to isolate in sufficient numbers and purity for functional analysis. To overcome these difficulties, we have developed a transgenic mouse model expressing enhanced green fluorescent protein in late distal convoluted tubules and connecting tubules. Enhanced green fluorescent protein expression was driven by the promoter for the transient receptor potential subfamily V, member 5. Confocal fluorescence microscopy allowed detection of enhanced green fluorescent protein in living isolated late distal convoluted tubules and connecting tubules and in the initial cortical collecting ducts. Enhanced green fluorescent protein expression was validated by double- and triple-fluorescence immunolabeling with specific tubule markers. Freshly isolated late distal convoluted tubules and connecting tubules increased their intracellular Ca²⁺ levels in response to the V2 receptor specific agonist, deamino-Cys,D-Arg⁸-vasopressin (2x10^-10 M) after 1 min of superfusion. In addition, both late distal convoluted tubules and connecting tubules displayed a concentration dependent intracellular Ca²⁺ response to 1,25-dihydroxyvitamin D₃ (range: 10^-10 to 10^-8 M). This suggests that 1,25-dihydroxyvitamin D₃ can act through a non-genomic signaling pathway in these tubules. In conclusion, the transgenic mouse model, expressing enhanced green fluorescent protein, is suitable for rapid isolation of viable late distal convoluted tubules, connecting tubules, and initial cortical collecting ducts, and provides an ideal tool for a more exhaustive functional characterization of these segments.