Hypokalemia is associated with increased ammoniagenesis and stimulation of net acid excretion by the kidney in both humans and experimental animals. The molecular mechanisms underlying these effects remain unknown. Toward this end, rats were placed in metabolic cages and fed a control or K⁺-deficient diet (KD) for up to 6 days. Rats subjected to KD showed normal acid-base status and serum electrolytes composition. Interestingly, urinary NH₄⁺ excretion increased significantly and correlated with a parallel decrease in urine K⁺ excretion in KD vs. control animals. Molecular studies showed a specific upregulation of the glutamine transporter SN1, which correlated with the upregulation of glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase. These effects occurred as early as day 2 of KD. Rats subjected to a combined KD and 280 mM NH₄Cl loading (to induce metabolic acidosis) for 2 days showed an additive increase in NH₄⁺ excretion along with an additive increment in the expression levels of ammoniagenic enzymes GA and GDH compared with KD or NH₄Cl loading alone. The incubation of cultured proximal tubule cells NRK 52E or LLC-PK₁ in low-K⁺ medium did not affect NH₄⁺ production and did not alter the expression of SN1, GA, or GDH in NRK cells. These results demonstrate that K⁺ deprivation stimulates ammoniagenesis through a coordinated upregulation of glutamine transporter SN1 and ammoniagenesis enzymes. This effect is developed before the onset of hypokalemia. The signaling pathway mediating these events is likely independent of KD-induced intracellular acidosis. Finally, the correlation between increased NH₄⁺ production and decreased K⁺ excretion indicate that NH₄⁺ synthesis and transport likely play an important role in renal K⁺ conservation during hypokalemia.