Ammonium chloride addition to drinking water is often used to induce metabolic acidosis (MA) in rodents but may also cause mild dehydration. Previous microarray screening of acidotic mouse kidneys showed upregulation of genes involved in renal water handling. Thus, we compared two protocols to induce metabolic acidosis in mice and rats: standard 0.28M NH₄Cl in drinking water or an equivalent amount of NH₄Cl in food. Both treatments induced MA in mice and rats. In rats, NH ₄Cl in water caused signs of dehydration, reduced mRNA abundance of the vasopression receptor 2 (V2R), increased protein abundance of the aquaporin water channels AQP2 and AQP3 and stimulated phosphorylation of AQP2 at residues Ser256 and Ser261. In contrast, NH₄Cl in food induced massive diuresis, decreased mRNA levels of V2R, AQP2, and AQP3, did not affect protein abundance of AQP2 and AQP3, and stimulated phosphorylation at Ser261 but not pSer256 of AQP2. In mice, NH₄Cl in drinking water stimulated urine concentration, increased AQP2 and V2R mRNA levels, and enhanced AQP2 and AQP3 protein expression with higher levels of AQP2 pSer256 and pSer261. Addition of NH₄Cl to food, stimulated diuresis, had no effect on mRNA levels of AQP2, AQP3, and V2R, and enhanced only AQP3 protein abundance whereas pSer256-AQP2 and pSer261-AQP2 remained unaltered. Similarly, AQP2 staining was more intense and luminal in kidney from mice with NH₄Cl in water but not in food. Pendrin, SNAT3 and PEPCK mRNA expression in mouse kidney were not affected by the route of N₄Cl application. Thus, addition of NH₄Cl to water or food causes MA but has differential effects on diuresis and expression of mRNAs and proteins related to renal water handling. Moreover, mice and rats respond differently to NH₄Cl loading, and increased water intake and diuresis may be a compensatory mechanism during MA. It may be necessary to consider these effects in planning and interpreting experiments of NH₄Cl supplementation to animals.