mIMCD-k2 cells are derived from the inner medullary collecting duct of a mouse and exhibit electrogenic sodium absorption and cAMP- and vasopressin (AVP)-stimulated electrogenic chloride secretion [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F347-F355, 1995; and N. L. Kizer, D. Vandorpe, B, Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F854-F861, 1995]. The purpose of the present study was to determine how peptide YY (PYY) affects electrogenic Na+ and Cl- current in mIMCD-k2 cells. Short-circuit currents (I(sc)) were measured across monolayers of mIMCD-k2 cells mounted in Ussing-type chambers. PYY did not alter baseline I(sc), nor did it alter I(sc) in chloride-free conditions, indicating no effect on electrogenic sodium transport. Baseline chloride current in these cells is low; therefore, chloride short-circuit current (I(sc)/(Cl)) was stimulated with AVP (10 nM) added to the basolateral surface and 10 μM amiloride added to the apical surface. Although apical applications of PYY had no effect, basolateral application of PYY caused attenuation of I(sc)/(Cl), with the maximal inhibitory dose (100 nM) causing 52 ± 1.3% inhibition (IC50 = 0.11 nM). Inhibition by PYY of I(sc)/(Cl) is mediated through the Y2 receptor subtype, as PYY-(3-36) was the only PYY analog tested that caused inhibition and was equipotent to PYY. Inhibition by PYY of I(sc)/(Cl) was abolished following incubation with pertussis toxin. We also show that PYY inhibits AVP-stimulated cAMP accumulation, with a maximal inhibitory dose (100 nM) causing a 38% ± 6% inhibition (IC50 = 0.16 nM), comparable to inhibition by PYY of I(sc)/(Cl). We conclude that PYY acts through either G(i) or G(o) to inhibit adenylate cyclase activity, leading to a decrease in AVP-stimulated chloride current.