Renal epithelia express multiple luminal and basolateral P2 receptors that mediate inhibition of water and electrolyte transport. Previously we identified luminal and basolateral P2Y₂ receptors in the medullary thick ascending limb (mTAL) as well as a basolateral P2X receptor. Transport studies investigating purinergic signaling in this nephron segment are currently pending. In this study, isolated, perfused mouse mTALs were used to electrically measure NaCl transport. The transepithelial voltage (V_te) and transepithelial resistance (R_te) were measured and via these the transepithelial NaCl absorption (I_sc) was calculated. Non-stimulated mTALs show a V_te: +7.12 ± 0.45 mV, an R_te: 6.62 ± 0.46 W cm² and an I_sc: 1104.87 ± 73.41 mA/cm² (n=12). Basolateral ATP (100mM) acutely and reversibly reduced the I_sc. After 2 minutes a maximal reduction amounted to ~25% (n=12). In the presence of ATP transport inhibition was sustained. Suramin, a P2 receptor antagonist, inhibited the ATP effect. Basolateral lead P2Y agonists like ADP, UTP or UDP had no effect on the NaCl transport. However, the non-specific P2X agonist 2-Methylthio ATP (100mM) was able to mimic the ATP effect. As this is a P2X₇ receptor agonist, we studied the P2X₇ knock-out mice mTALs, which still presented a significant ATP effect. These data indicate that basolateral ATP exerts a significant inhibition of NaCl absorption in mouse mTAL. Pharmacology uncovered a P2X receptor-mediated mechanism. Further studies are needed to clarify the identity of the P2X receptor involved in this NaCl transport regulation.