To determine the effect of glucose availability on glutamine metabolism, glutamine kinetics were assessed under conditions of hyperglycemia resulting from 1) intravenous infusion of 7.5% dextrose in healthy adults and 2) insulin deficiency in young adults with insulin-dependent diabetes mellitus (IDDM). Eight healthy adults and five young adults with IDDM were studied in the postabsorptive state by use of a primed continuous infusion of D-[U-¹⁴C]glucose, L-[5,5,5-²H₃]leucine, and L-[3,4-¹³C]glutamine. Whether resulting from insulin deficiency or dextrose infusion, the rise in plasma glucose was associated with increased glucose turnover (23.5 ± 0.7 vs. 12.9 ± 0.3 µmol · kg¹ · min¹, P < 0.01 and 20.9 ± 2.5 vs. 12.8 ± 0.4 µmol · kg¹ · min¹, P = 0.03, in health and IDDM, respectively). In both cases, high blood glucose failed to alter glutamine appearance rate (R_a) into plasma [298 ± 9 vs. 312 ± 14 µmol · kg¹ · h¹, not significant (NS) and 309 ± 23 vs 296 ± 26 µmol · kg¹ · h¹, NS, in health and IDDM, respectively] and the estimated fraction of glutamine R_a arising from de novo synthesis (210 ± 7 vs. 217 ± 10 µmol · kg¹ · h¹, NS and 210 ± 16 vs. 207 ± 21 µmol · kg¹ · h¹, NS, in health and IDDM, respectively). When compared with the euglycemic day, the apparent contribution of glucose to glutamine carbon skeleton increased when high plasma glucose resulted from intravenous dextrose infusion in healthy volunteers (10 ± 0.8 vs. 4.8 ± 0.3%, P < 0.01) but failed to do so when hyperglycemia resulted from insulin deficiency in IDDM. We conclude that 1) the contribution of glucose to the estimated rate of glutamine de novo synthesis does not increase when elevation of plasma glucose results from insulin deficiency, and 2) the transfer of carbon from glucose to glutamine may depend on insulin availability.