Abstract The intracellular pH, pH i, of identified neurones of the central nervous system of the leech Hirudo medicinalis L. was measured with double-barrelled neutral carrier pH-sensitive microelectrodes. The active regulation of pH i of these neurons is due to amiloride-sensitive Na H exchange and hence requires extracellular Na, Na o. We have measured a decrease of pH i following the removal of Na o. The rate of intracellular acidification in Na-free saline was similar to that in the presence of 2 mM amiloride suggesting that the acidification was due to inhibition of the Na H exchange. The rate of intracellular acidification depended on the Na substitute chosen; it was 0.02 ± 0.005 pH units/min (±S.D., n = 17) when Na was replaced by N-methyl- d-glucamine. A similar rate of acidification occurred with tris-hydroxymethyl-aminomethane (Tris) while the rate of acidification was higher with bis-2-hydroxymethyl-dimethyl-ammonium (BDA, 0.033 ± 0.016 pH units/min (±S.D., n = 7) and tetramethylammonium TMA, 0.046 ± 0.017 pH units/min ( n = 3) as Na substitutes. A high, non-linear rate of intracellular acidification was observed, when Li, K or choline were used as Na substitute. The recovery of pH i from acidification upon readdition of Na o was fast, only when Li had replaced Na was the pH i recovery considerably delayed. In conclusion, in all experiments using different Na substitutes the removal of Na o caused a ssubstantial intracellular acidification presumably due to inhibition of Na H exchange. These changes in pH i might be relevant for results obtained by experiments in which Na-free solutions are used.