Extracellular nucleotides regulate NaCl transport in some epithelia. However, the effects of nucleotide agonists on NaCl transport in the renal inner medullary collecting duct (IMCD) are not known. The objective of this study was to determine whether ATP and related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited Na+ absorption [measured via Na+ short-circuit current (I(sc)/Na))] and stimulated Cl- secretion [measured via Cl- short-circuit current (I(sc)/Cl))]. Using selective P2 agonists, we report that P2X and P2Y purinoceptors regulate I(sc)/Na) and I(sc)/(Cl). By RT-PCR, two P2X receptor channels (P2X3, P2X4) and two P2Y G protein-coupled receptors (P2Y1, P2Y2) were identified. Functional localization of P2 purinoceptors suggest that I(sc)/(Cl) is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas I(sc)/(Na) is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. Together, we conclude that nucleotide agonists inhibit I(sc)/(Na) across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane, whereas extracellular nucleotides stimulate I(sc)/(Cl) through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.