Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na⁺ absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current (I_sc) and ²²Na⁺ flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive I_sc and ²²Na⁺ absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive I_sc had an IC₅₀ value of 20-30 µM, with the maximum inhibition being ~50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg²⁺ and Ca²⁺ from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca²⁺ did not affect the percent inhibition of amiloride-sensitive I_sc induced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive I_sc, this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na⁺ absorption. This effect was probably mediated by a P2Y₂ receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca²⁺ concentration may not be necessary for this process.