Glutamine is the most abundant amino acid in the body and is extensively taken up in gut and liver in healthy humans. To determine whether glucocorticosteroids alter splanchnic glutamine metabolism, the effect of prednisone was assessed in healthy volunteers using isotope tracer methods. Two groups of healthy adults received 5-hr intravenous infusions of L-[1-¹⁴C]leucine and L-[²H₅]glutamine, along with q. 20 min oral sips of tracer doses of L-[1-¹³C]glutamine in the fasting state, either: (a) at baseline (control group; n=6), or (b) after a 6-day course of 0.8 mg.kg^-1.d^-1 prednisone (prednisone group; n=8). Leucine and glutamine appearance rates (Ra) were determined from plasma [1-¹⁴C]-ketoisocaproate and [²H₅]-glutamine, respectively; and leucine and glutamine oxidation from breath ¹⁴CO₂ and ¹³CO₂, respectively. Splanchnic glutamine extraction was estimated by the fraction of orally administered ¹³C-glutamine that failed to appear into systemic blood. Prednisone treatment: 1) did not affect leucine rate of appearance (Ra) or leucine oxidation; 2) increased plasma glutamine Ra, mostly due to enhanced glutamine de novo synthesis (median±IQ, 412±61 vs. 280±190 µmol.kg^-1.h^-1, P=0.003); and 3) increased the fraction of orally administered glutamine undergoing extraction in the splanchnic territory (64±6 vs. 42±12 %, P<0.05), without any change in the fraction of glutamine oxidized (mean±SE, 75±4 vs. 77±4 % %, NS). We conclude that high dose glucocorticosteriods increase in splanchnic bed the glutamine requirements. The role of such changes in patients receiving chronic corticoid treatment for inflammatory diseases or suffering from severe illness remain to be determined.