Ammonium is the most common N source for yeast fermentations. Although its transport and assimilation mechanisms are well documented, there have been only a few attempts to measure the in vivo intracellular concentration of ammonium and assess its impact on gene expression. Using an isotope dilution mass spectrometry (IDMS)-based method, we were able to measure the intracellular ammonium concentration in N-limited aerobic chemostat cultivations using three different N sources (ammonium, urea, and glutamate) at the same growth rate (0.05 h-1). The experimental results suggest that, at this growth rate, a similar concentration of intracellular (IC) ammonium, about 3.6 mmol NH4 +/literIC, is required to supply the reactions in the central N metabolism, independent of the N source. Based on the experimental results and different assumptions, the vacuolar and cytosolic ammonium concentrations were estimated. Furthermore, we identified a futile cycle caused by NH3 leakage into the extracellular space, which can cost up to 30% of the ATP production of the cell under N-limited conditions, and a futile redox cycle between Gdh1 and Gdh2 reactions. Finally, using shotgun proteomics with protein expression determined relative to a labeled reference, differences between the various environmental conditions were identified and correlated with previously identified N compound-sensing mechanisms.