Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pHcyt) and vacuolar pH (pHvac) values of 7.6 and 6.2, respectively, when the extracellular pH (pHex) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a ΔpH over the cytoplasmic membrane of 6.6–6.7 was reached (pHex 0.7–0.8). Maintenance of these large pH differences was possible without increased respiration compared to pHex 5.8. Perfusion in the presence of various hexoses and pentoses (pHex 5.8) revealed that the magnitude of ΔpH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger ΔpH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 µm CCCP), transient (2 µm CCCP) or permanent (10 µm CCCP) collapse of the vacuolar membrane ΔpH. Nonlethal levels of the metabolic inhibitor azide (N3–, 0.1 mm) caused a transient decrease in pHcyt that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pHex (1.8) and a high acid-secreting capacity, pHcyt and pHvac values were still well maintained (pH 7.5 and 6.4, respectively).