Acute and chronic homeostatic pH regulation is critical for the maintenance of optimal cellular function. Renal mechanisms dominate global pH regulation over longer time frames, and rapid adjustments in ventilation compensate for acute pH and CO 2 changes. Ventilatory CO 2 and pH chemoreflexes are primarily determined by brain chemoreceptors with intrinsic pH sensitivity likely driven by K+ channels. Here, we studied acute and chronic pH regulation in Kcnj16 mutant Dahl salt-sensitive (SS Kcnj16−/−) rats; Kcnj16 encodes the pH-sensitive inwardly rectifying K+ 5.1 (Kir5. 1) channel. SS Kcnj16−/− rats hyperventilated at rest, likely compensating for a chronic metabolic acidosis. Despite their resting hyperventilation, SS Kcnj16−/− rats showed up to 45% reduction in the ventilatory response to graded hypercapnic acidosis vs. controls. SS Kcnj16−/− rats chronically treated with bicarbonate or the carbonic anhydrase inhibitor hydrochlorothiazide had partial restoration of arterial pH, but there was a further reduction in the ventilatory response to hypercapnic acidosis. SS Kcnj16−/− rats also had a nearly absent hypoxic ventilatory response, suggesting major contributions of Kir5. 1 to O 2-and CO 2-dependent chemoreflexes. Although previous studies demonstrated beneficial effects of a high-K+ diet (HKD) on cardiorenal phenotypes in SS Kcnj16−/− rats, HKD failed to restore the observed ventilatory phenotypes. We conclude that Kir5. 1 is a key regulator of renal H+ handling and essential for acute and chronic regulation of arterial pH as determinants of the ventilatory CO 2 chemoreflex.—Puissant, MM, Muere, C., Levchenko, V., Manis, AD, Martino, P., Forster, HV, Palygin, O., Staruschenko, A., Hodges, MR Genetic mutation of Kcnj16 identifies Kir5. 1-containing channels as key regulators of acute and chronic pH homeostasis.