The short term effect of heavy water (2H2O) in intracellular pH (pHi) and phosphatidylcholine (PtdCho) turnover have been studied by 31P NMR spectroscopy in the perfused mouse liver metabolizing alanine. Hepatic pHi decreased from 7.19 +/- 0.01 (n = 10) to 7.01 +/- 0.03 (n = 4) after the addition of 6 mM alanine to Krebs Ringer bicarbonate (KRB) perfusion medium. Replacement of 50% of the KRB water with 2H2O during alanine perfusion inhibited the intracellular acidification induced by alanine and caused i) a decrease in the hepatic content of PtdCho, and ii) increases in phosphocholine and glycerophosphocholine, respectively. Amiloride (1 mM) of 5-(N-ethyl-N-isopropyl)-amiloride (10 microM), two previously reported inhibitors of the Na+/H+ exchangers, mimicked the effects produced by 2H2O on pHi and PtdCho turnover. Replacement of 50% of the KRB water with 2H2O or the addition of 1mM amiloride to KRB only, did not modify pHi nor increase the levels of phosphocholine of glycerophosphocholine. Thus, the observed increases are the result of alanine perfusion in the presence of 2H2O or amiloride. These results suggest that 2H2O behaves similarly to previously reported inhibitors of Na+/H+ exchange, disclosing also a novel role for PtdCho metabolism in the regulation on hepatic pHi.