E xtracellular nucleotides are important regulators of epithelial ion transport. Here we investigated nucleotide-mediated effects on colonic NaCl secretion and the signal transduction mechanisms involved. Basolateral UDP induced a sustained activation of Cl^– secretion, which was completely inhibited by 293B, a specific inhibitor of cAMP-stimulated basolateral KCNQ1/KCNE3 K⁺ channels. We therefore speculated that a basolateral P2Y₆ receptor could increase cAMP. Indeed UDP elevated cAMP in isolated crypts. We identified an epithelial P2Y₆ receptor using crypt [Ca²⁺]_i measurements, RT-PCR, and immunohistochemistry. To investigate whether the rat P2Y₆elevates cAMP, we coexpressed the P2Y₁ or P2Y₆ receptor together with the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel in Xenopus oocytes. A two-electrode voltage clamp was used to monitor nucleotide-induced Cl^– currents. In oocytes expressing the P2Y₁ receptor, ATP transiently activated the endogenous Ca²⁺-activated Cl^– current, but not CFTR. In contrast, in oocytes expressing the P2Y₆receptor, UDP transiently activated the Ca²⁺-activated Cl^– current and subsequently CFTR. CFTR Cl^– currents were identified by their halide conductance sequence. In summary we find a basolateral P2Y₆ receptor in colonic epithelial cells stimulating sustained NaCl secretion by way of a synergistic increase of [Ca²⁺]_i and cAMP. In support of these data P2Y₆ receptor stimulation differentially activates CFTR in Xenopus oocytes.