Extracellular ATP is an important regulator of renal tubular transport. Recently, we found that basolateral ATP markedly inhibits NaCl absorption in mouse medullary thick ascending limb (mTAL). A basolateral P2X receptor was found to be responsible for this effect. However, the underlying mechanism of this ATP stimulated transport inhibition in mTAL is unclear. To investigate this we measured pH_i in single perfused mouse mTALs using the fluorescent ratiometric dye BCECF. Interestingly, basolateral ATP (100 mM) caused a prominent and reversible alkalization of mTAL, with an average pH_i increase of 0.14 ± 0.01 (n=12). This effect was completely abolished in presence of the P2X receptor antagonist oxidized-ATP (50 mM). Typically, GPCR activation of tubular epithelial cells that trigger an increase of [Ca²⁺]_i lead to a significant acidification. This was also observed in this study, when mTAL P2Y₂ receptors or CaSR were stimulated with either 100 mM UTP or 5 mM Ca²⁺. This study therefore describes the surprising finding of a basolateral P2X receptor triggered alkalinzation in isolated mouse mTAL. These data may provide the basis to understand the signaling mechanism responsible for the pronounced ATP-induced transport-inhibition in mouse mTAL.