Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors, which mediate significant inhibition of solute absorption. Recently, we identified several P2 receptors in the medullary thick ascending limb (mTAL) including luminal and basolateral P2Y(2) receptors (Jensen ME, Odgaard E, Christensen MH, Praetorius HA, Leipziger J. J Am Soc Nephrol 18: 2062-2070, 2007). In addition, we found evidence for a basolateral P2X receptor. Here, we investigate the effect of basolateral ATP on NaCl absorption in isolated, perfused mouse mTALs using the electrical measurement of equivalent short-circuit current (I'(sc)). Nonstimulated mTALs transported at a rate of 1,197 ± 104 μA/cm(2) (n = 10), which was completely blockable with luminal furosemide (100 μM). Basolateral ATP (100 μM) acutely (1 min) and reversibly reduced the absorptive I'(sc). After 2 min, the reduction amounted to 24.4 ± 4.0% (n = 10). The nonselective P2 receptor antagonist suramin blocked the effect. P2Y receptors were found not to be involved in this effect. The P2X receptor agonist 2-methylthio ATP mimicked the ATP effect, and the P2X receptor antagonist periodate-oxidized ATP blocked it. In P2X(7)(-/-) mice, the ATP effect remained unaltered. In contrast, in P2X(4)(-/-) mice the ATP-induced inhibition of transport was reduced. A comprehensive molecular search identified P2X(4), P2X(5), and P2X(1) receptor subunit mRNA in isolated mouse mTALs. These data define that basolateral ATP exerts a significant inhibition of Na(+) absorption in mouse mTAL. Pharmacological, molecular, and knockout mouse data identify a role for the P2X(4) receptor. We suggest that other P2X subunits like P2X(5) are part of the P2X receptor complex. These data provide the novel perspective that an ionotropic receptor and thus a nonselective cation channel causes transport inhibition in an intact renal epithelium.