The aims of this work were to establish whether glutamine infusion via alanyl-glutamine dipeptide provides effective therapy against muscle atrophy from glucorticoids and whether the glucocorticoid induction of glutamine synthetase (GS) is downregulated by dipeptide supplementation. Rats were given hydrocortisone 21-acetate or the dosing vehicle and were infused with alanine (AA) or alanyl-glutamine (AG) at the same concentrations and rates (1.15 mumol.min-1.100 g body wt-1, 0.75 ml/h) for 7 days. Compared with AA infusion in hormone-treated animals, AG infusion prevented total body and fast-twitch muscle mass losses by over 70%. Glucocorticoid treatment did not reduce muscle glutamine levels. Higher serum glutamine was found in the AG-infused (1.72 +/- 0.28 mumol/ml) compared with the AA-infused group (1.32 +/- 0.06 mumol/ml), but muscle glutamine concentrations were not elevated by AG infusion. Following glucocorticoid injections, GS enzyme activity was increased by two- to threefold in plantaris, fast-twitch white (superficial quadriceps), and fast-twitch red (deep quadriceps) muscle/fiber types of the AA group. Similarly, GS mRNA was elevated by 3.3- to 4.1-fold in these same muscles of hormone-treated, AA-infused rats. AG infusion diminished glucocorticoid effects on GS enzyme activity to 52-65% and on GS mRNA to 31-37% of the values with AA infusion. These results provide firsthand evidence of atrophy prevention from a catabolic state using glutamine in dipeptide form. Despite higher serum and muscle alanine levels with AA infusion than with AG infusion, alanine alone is not a sufficient stimulus to counteract muscle atrophy. The AG-induced muscle sparing is accompanied by diminished expression of a glucocorticoid-inducible gene in skeletal muscle. However, glutamine regulation of GS appears complex and may involve more regulators than muscle glutamine concentration alone.