In root hairs of Medicago sativa, pH-sensitive microelectrodes have been applied to study cytoplasmic pH-regulation. To inhibitors like oligomycin, antimycin A, cyanide and the exchange of O2 for N2, the root hairs respond with a distinct cytoplasmic acidification. Whereas the cytoplasmic pH under aerobic conditions rests at 7.28 ± 0.11 SE (n = 168), under conditions of (chemical) anoxia the cytoplasmic pH is shifted to a stable, well-regulated 6.78 ± 0.08 SE (n = 81). Once this pH is attained in the presence of one inhibitor, addition of another has no effect. 2-deoxyglucose and N-acetylglucosamine, both inhibitors of glycolysis at the hexokinase level, increase cytoplasmic pH by about 0.3 pH units, as do glucogenic amino acids. It is suggested that aerobic energy metabolism does not contribute to acidosis of these cells. Since pH-shift and pump deactivation can be separated by using poor respiratory inhibitors, it is concluded that the switch from ‘aerobic’ to ‘anaerobic’ pH is not correlated with proton pump activity. Inversely, since cytoplasmic pH neither responds to pump activation by FC with alkalinization, nor to pump deactivation by cyanide with acidification, it is also concluded that changes in pump activity do not affect cytoplasmic pH.