Inwardly rectifying potassium channels play essential roles in renal physiology across phyla. Barium-sensitive K(+) conductances are found on the basolateral membrane of a variety of insect Malpighian (renal) tubules, including Drosophila melanogaster. We found that barium decreases the lumen-positive transepithelial potential difference in isolated perfused Drosophila tubules and decreases fluid secretion and transepithelial K(+) flux. In those insect species in which it has been studied, transcripts from multiple genes encoding inwardly rectifying K(+) channels are expressed in the renal (Malpighian) tubule. In Drosophila melanogaster, this includes transcripts of the Irk1, Irk2, and Irk3 genes. The role of each of these gene products in renal tubule function is unknown. We found that simultaneous knockdown of Irk1 and Irk2 in the principal cell of the fly tubule decreases transepithelial K(+) flux, with no additive effect of Irk3 knockdown, and decreases barium sensitivity of transepithelial K(+) flux by ∼50%. Knockdown of any of the three inwardly rectifying K(+) channels individually has no effect, nor does knocking down Irk3 simultaneously with Irk1 or Irk2. Irk1/Irk2 principal cell double-knockdown tubules remain sensitive to the kaliuretic effect of cAMP. Inhibition of the Na(+)/K(+)-ATPase with ouabain and Irk1/Irk2 double knockdown have additive effects on K(+) flux, and 75% of transepithelial K(+) transport is due to Irk1/Irk2 or ouabain-sensitive pathways. In conclusion, Irk1 and Irk2 play redundant roles in transepithelial ion transport in the Drosophila melanogaster renal tubule and are additive to Na(+)/K(+)-ATPase-dependent pathways.