Cl⁻-sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive Na⁺-Cl⁻ cotransporter (NCC) in the distal convoluted tubule (DCT). Cl⁻ enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl⁻ channel ClC-K2 or K⁺-Cl⁻ cotransporter (KCC). While KCC is electroneutral, Cl⁻ exit via ClC-K2 is electrogenic. Therefore, an alteration in DCT basolateral K⁺ channel activity is expected to influence Cl⁻ movement across the basolateral membrane. Although a role for intracellular Cl⁻ in the regulation of WNK and NCC has been established, intracellular Cl⁻ concentrations ([Cl⁻]_i) have not been directly measured in the mammalian DCT. Therefore, to measure [Cl⁻]_i in DCT cells, we generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl⁻ fluorescent imaging in the isolated DCT. Basal measurements indicated that the mean [Cl⁻]_i was ~7 mM. Stimulation of Cl⁻ exit with low-Cl⁻ hypotonic solutions decreased [Cl⁻]_i, whereas inhibition of KCC by DIOA or inhibition of ClC-K2 by NPPB increased [Cl⁻]_i, suggesting roles for both KCC and ClC-K2 in the modulation of [Cl⁻]_i . Blockade of basolateral K⁺ channels (Kir4.1/5.1) with barium significantly increased [Cl⁻]_i. Finally, a decrease in extracellular K⁺ concentration transiently decreased [Cl⁻]_i, whereas raising extracellular K⁺ transiently increased [Cl⁻]_i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl⁻]_i. We conclude that the alteration in ClC-K2, KCC, and Kir4.1/5.1 activity influences [Cl⁻]_i in the DCT.