Glucocorticoids are involved in many aspects of regulation of acid-base homeostasis, including the stimulation of renal ammoniagenesis during chronic metabolic acidosis. Plasma glutamine is the principal substrate for ammoniagenesis under these conditions. Expression of the System N glutamine transporter SNAT3 is increased in the renal proximal tubules during acidosis. In vivo studies in rats using 1) sham and adrenalectomized rats, 2) the glucocorticoid receptor antagonist RU486, and 3) dexamethasone treatment demonstrated involvement of glucocorticoids in regulation of SNAT3 expression. Adrenalectomy attenuated the acidosis-induced increase in renal cortical SNAT3 mRNA approximately 40% and treatment with dexamethasone (1 mg/kg/day, sc) partially reversed this effect. RU486 also blunted the acidosis-induced increase in SNAT3 expression approximately 50%. Chronic dexamethasone treatment (0.1 mg/day, sc, 6 days) of normal rats slightly increased SNAT3 expression. In all cases, renal glutamine arteriovenous difference mirrored SNAT3 expression and activity in the proximal tubules, suggesting that glutamine uptake during acidosis is regulated by SNAT3. These studies indicate that glucocorticoids regulate acid-base homeostasis during metabolic acidosis in part by regulating expression of the System N transporter SNAT3.