Glucose metabolism in free-swimming fasted and fed seabass was studied using deuterated water ( 2 H 2 O). After transfer to seawater enriched with 4.9% 2 H 2 O for 6-h or for 72-h, positional and mole percent enrichment (MPE) of plasma glucose and water were quantified by 2 H NMR and ESI-MS/MS. Plasma water 2 H-enrichment reached that of seawater within 6h. In both fasted and fed fish, plasma glucose MPE increased asymptotically attaining ~55% of plasma water enrichment by 72h. The distribution of 2 H-enrichment between the different glucose positions was relatively uniform. The gluconeogenic contribution to glucose that was synthesized during 2 H 2 O administration was estimated from the ratio of position 5 and 2 glucose enrichments. For both fed and fasted fish, gluconeogenesis accounted for 98±1% of the glucose that was produced during the 72-h 2 H 2 O administration period. For fasted fish, gluconeogenic contributions measured after 6h were identical to 72-h values (94±3%). For fed fish, the apparent gluconeogenic contribution at 6-h was significantly lower compared to 72-h (79±5% versus 98±1%, p<0.05). This may reflect a brief augmentation of gluconeogenic flux by glycogenolysis after feeding and/or selective enrichment of plasma glucose position 2 via futile glucose-glucose-6-phosphate cycling.