Tritiated water and radioactive tracers have been used to monitor glucose production by primary cultures of hepatocytes. More recently, 3H2O has been replaced for by 2H2O in 'in vivo' studies addressed at the evaluation of the relative contribution of gluconeogenesis to total glucose production. In this work, the possibility of using 2H2O to determine the ratio between the glucogenic flux and the overall flux through glucose 6-phosphate in isolated liver cells in vitro was evaluated. For this purpose, hepatocytes from either fasted or fed rats were incubated with a medium containing 6, 12 and 25% of 2H2O in the presence of either 2 or 20 mM pyruvate. Isotopomer analysis of six different mass clusters (m/z 328, 314, 242, 212, 187 and 145) was carried out by gas chromatography/mass spectrometry (GC/MS) of glucose aldonitrile pentaacetate. For each cluster, ions at m/z +1, +2, +3 and +4 were monitored. From the combination of different clusters the enrichment at C-6 and C-2 of glucose was computed and the C-6/C-2 ratio was considered to represent the contribution of gluconeogenesis to total glucose production, as suggested previously. Based on the results obtained, conditions selected to be optimum for the use of the method in studies on the modulation of gluconeogenesis were as follows: incubation of hepatocytes with 20 mM pyruvate in 12% 2H2O followed GC/electron ionization MS analysis of the clusters of ions at m/z 328, 314 and 187 of the glucose derivative to calculate enrichment at the C-2 and C-6 positions of glucose.