The use of 2H2O in estimating gluconeogenesis' contribution to glucose production (%GNG) was examined during progressive fasting in three groups of healthy subjects. One group (n = 3) ingested 2H2O to a body water enrichment of approximately 0.35% 5 h into the fast. %GNG was determined at 2-h intervals from the ratio of the enrichments of the hydrogens at C-5 and C-2 of blood glucose, assayed in hexamethylenetetramine. %GNG increased from 40 +/- 8% at 10 h to 93 +/- 6% at 42 h. Another group ingested 2H2O over 2.25 h, beginning at 11 h (n = 7) and 19 h (n = 7) to achieve approximately 0.5% water enrichment. Enrichment in plasma water and at C-2 reached steady state approximately 1 h after completion of 2H2O ingestion. The C-5-to-C-2 ratio reached steady state by the completion of 2H2O ingestion. %GNG was 54 +/- 2% at 14 h and 64 +/- 2% at 22 h. A 3-h [6,6-2H2]glucose infusion was also begun to estimate glucose production from enrichments at C-6, again in hexamethylenetetramine. Glucose produced by gluconeogenesis was 0.99 +/- 0.06 mg.kg-1.min-1 at both 14 and 22 h. In a third group (n = 3) %GNG reached steady state approximately 2 h after 2H2O ingestion to only approximately 0.25% enrichment. In conclusion, %GNG by 2 h after 2H2O ingestion and glucose production using [6,6-2H2]glucose infusion, begun together, can be determined from hydrogen enrichments at blood glucose C-2, C-5, and C-6. %GNG increases gradually from the postabsorptive state to 42 h of fasting, without apparent change in the quantity of glucose produced by gluconeogenesis at 14 and 22 h.