Modulation of the epithelial Na+ channel (ENaC) activity in the collecting duct (CD) is an important mechanism for normal Na+ homeostasis. ENaC activity is inversely related to dietary Na+ intake, in part, due to inhibitory paracrine purinergic regulation. Evidence suggests that H+,K+-ATPase activity in the CD also influences Na+ excretion. We hypothesized that renal H+,K+-ATPases affect Na+ reabsorption by the CD by modulating ENaC activity. ENaC activity in HKα1 H+,K+-ATPase knockout (HKα1-/-) mice was uncoupled from Na+ intake. ENaC activity on a high Na+ diet was greater in the HKα1-/- mice compared to WT mice. Moreover, dietary Na+ content did not modulate ENaC activity in the HKα1 /- mice as it did in WT mice. Purinergic regulation of ENaC was abnormal in HKα1-/- mice. In contrast to WT where urinary [ATP] was proportional to dietary Na+ intake, urinary [ATP] did not increase in response to a high Na+ diet in the HKα1-/- and was significantly lower than in the WT . HKα1-/- mice fed a high Na+ diet had greater Na+ retention compared to WT mice and had an impaired dipsogenic response. These results suggest an important role for the HKα1 subunit in the regulation of purinergic signaling in the CD. They are also consistent with HKα1-containing H+,K+-ATPases as important components for the proper regulation of Na+ balance and the dipsogenic response to a high salt diet. Such observations suggest a previously unrecognized element in Na+ regulation in the CD.