ROMK-deficient (Romk^-/-) mice exhibit polyuria, natriuresis and kaliuresis similar to individuals with Type II Bartters form of hyperprostaglandin E syndrome (HPS; antenatal Bartters syndrome). ROMK-deficient (Romk^-/-) mice exhibit polyuria, natriuresis and kaliuresis similar to individuals with Type II Bartters form of hyperprostaglandin E syndrome (HPS; antenatal Bartters syndrome). In the present study, we utilized both metabolic and clearance studies to define the contributions of specific distal nephron segments to the renal salt wasting in these mice. The effects of furosemide, hydrochlorothiazide, and benzamil on urinary Na⁺ and K⁺ excretion in both wild type (Romk^+/+) and Romk^-/- mice were used to assess and compare salt transport by the NKCC2-expressing thick ascending limb (TAL), the NCC-expressing distal convoluted tubule (DCT1/DCT2) and the ENaC-expressing connecting segment (CNT) and collecting duct (CD), respectively. Whole kidney GFR was reduced by 47% in Romk^-/- mice. Furosemide-induced increments in FENa, ENa, FEK and EK were significantly blunted in Romk^-/- mice, consistent with a major salt transport defect in the TAL. In contrast, hydrochlorothiazide produced an exaggerated natriuresis in Romk^-/- mice indicating upregulation of salt absorption by the DCT. Benzamil resulted in a similar increment in ENa in both Romk^-/- and Romk^+/+ indicating no significant upregulation of Na transport by ENaC in ROMK-null mice. Moreover, hydrochlorothiazide increased FEK in Romk^-/- mice, confirming our recent observation that maxi-K channels contribute to distal K secretion in the absence on ROMK.