The interaction of glutamine availability and glucose homeostasis during and after exercise were investigated, measuring whole-body glucose kinetics with [3-³H]glucose and net organ balances of glucose and amino acids (AA) during basal, exercise, and postexercise hyperinsulinemic-euglycemic clamp periods in six multicatheterized dogs. Dogs were studied twice in random treatment order: once with glutamine (12 µmol/kg/min; GLN) and once with saline (CON) infused intravenously during and after exercise. Plasma glucose fell 7 mg/dl with exercise in CON (p<0.05), but did not fall with GLN. GLN further stimulated whole-body glucose production and utilization an additional 24% above a normal exercise response (p<0.05). Net hepatic uptake of glutamine and alanine was greater with GLN than CON during exercise (p<0.05). Net hepatic glucose output was increased 7-fold during exercise with GLN (p<0.05), but not with CON. Net hindlimb glucose uptake was increased similarly during exercise in both groups (p<0.05). During the post-exercise hyperinsulinemic-euglycemic period, glucose production decreased to near zero with CON, but did not decrease below basal levels with GLN. GLN increased glucose utilization by 16% compared to CON after exercise (p<0.05). Furthermore, net hindlimb glucose uptake in the post-exercise period was increased ~2- fold versus basal with GLN (p<0.05), but not with CON. Net hepatic uptake of glutamine during the post-exercise period was three-fold greater for GLN than CON (p<0.05). In conclusion, glutamine availability modulates glucose homeostasis during and after exercise, which may have implications for post-exercise recovery.