Background: Microbial production of nitrogen containing compounds requires a high uptake flux and assimilation of the N-source (commonly ammonium), which is generally coupled with ATP consumption and negatively influences the product yield. In the industrial workhorse Saccharomyces cerevisiae, ammonium (NH₄ ⁺) uptake is facilitated by ammonium permeases (Mep1, Mep2 and Mep3), which transport the NH₄ ⁺ ion, resulting in ATP expenditure to maintain the intracellular charge balance and pH by proton export using the plasma membrane-bound H⁺-ATPase. Results: To decrease the ATP costs for nitrogen assimilation, the Mep genes were removed, resulting in a strain unable to uptake the NH₄ ⁺ ion. Subsequent analysis revealed that growth of this Δmep strain was dependent on the extracellular NH₃ concentrations. Metabolomic analysis revealed a significantly higher intracellular NH_X concentration (3.3-fold) in the Δmep strain than in the reference strain. Further proteomic analysis revealed significant up-regulation of vacuolar proteases and genes involved in various stress responses. Conclusions: Our results suggest that the uncharged species, NH₃, is able to diffuse into the cell. The measured intracellular/extracellular NH_X ratios under aerobic nitrogen-limiting conditions were consistent with this hypothesis when NH_x compartmentalization was considered. On the other hand, proteomic analysis indicated a more pronounced N-starvation stress response in the Δmep strain than in the reference strain, which suggests that the lower biomass yield of the Δmep strain was related to higher turnover rates of biomass components.