Isotope effects on the central carbon metabolism due to the addition of variable amounts of D2O (0 to 70%) were investigated with biosynthetically directed fractional 13C-labeling for Escherichia coli BL21(DE3) cells during exponential growth on a M9 minimal medium containing a mixture of 70% unlabeled and 30% uniformly 13C-labeled glucose as the sole carbon source. The resulting 13C-labeling patterns in the amino acids were analysed by two-dimensional [13C,1H]-correlation spectroscopy. With the aforementioned growth conditions, higher D2O contents resulted in an increase of the anaplerotic supply of the tricarboxylic acid cycle via carboxylation of phosphoenolpyruvate when compared to the influx of acetyl-CoA. Furthermore, the addition of D2O affected the C1 metabolic pathways that involve Ser and Gly. Otherwise the E. coli cells showed identical topologies of the active biosynthetic pathways in H2O and at elevated D2O contents, and the metabolic flux ratios characterizing glycolysis and the pentose phosphate pathway were not measurably affected by the addition of D2O. Cells that had been adapted for growth in D2O exhibited the same response to the presence of D2O in the nutrient medium as non-adapted cells. Implications of these data for the preparation of recombinant deuterated proteins for NMR studies are discussed.