We have previously investigated, in studies of rat distal colonic mucosa, the effect of ATP added to the basolateral side on ion transport and [Ca²⁺]_i. It was demonstrated that ATP acts via a P2Y₁ receptor to increase [Ca²⁺]_i and NaCl secretion. In the present study we investigated the effect of luminally added nucleotides (ATP, UTP) on transepithelial voltage (V _te) and resistance (R _te) in Ussing chamber experiments on rat distal colonic mucosa. Both nucleotides induced a rapid and transient (within 30 s) change of V _te to lumen-positive values (resting V _te: –2±1 mV; peak V _te after 100 µmol/l ATP: +2.4±1.1 mV) and a decrease of R _te from 89.9±10.3 to 83.8±9.1 Ωcm² (n=10). Similar values were obtained with luminal UTP (n=15). The estimated EC₅₀ values for both nucleotides were approximately 6 µmol/l. The ATP-induced V _te effect was nearly completely sensitive to Ba²⁺. Addition of the K⁺ channel blocker Ba²⁺ (1 mmol/l) to the luminal solution reversibly inhibited 77±4% (n=5) of the ATP-induced V _te effect. Experiments to identify the respective P2 receptor subtype revealed the following rank order of potency at 500 µmol/l agonist: UTP≥ATP>>2-methylthio-ATP=ADP>>adenosine> AMP>β,γ-methylene-ATP (n=5). This closely resembles the published rank order for the P2Y₂ receptor. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique P2Y₂ receptor-specific mRNA was detected in total RNA extracted from isolated crypts. In summary these data indicate that luminal ATP and UTP act via a P2Y₂ receptor in the luminal membrane of colonic mucosa to elicit a transient K⁺ secretion.