Dexras1 is a novel GTP-binding protein that shares structural similarity with the Ras family of small molecular weight GTPases and is strongly and rapidly induced during treatment with dexamethasone. The function of Dexras1 and its contribution to glucocorticoid-dependent signaling in the corticotroph cell are unknown. The present study was undertaken to examine the potential role of Dexras1 in the regulation of peptide hormone secretion in the AtT-20 corticotroph cell line. To determine the effects of Dexras1 expressed independently of glucocorticoid treatment, expression plasmids for wild-type and constitutively active mutant Dexras1 proteins were cotransfected with human GH (hGH), which provides an ectopic marker for the stimulus-coupled secretory pathway. GTP binding properties and the GTP to GDP ratio of wild-type and mutant Dexras1 proteins were examined in transiently transfected AtT-20 and COS-7 cells. Stimulated and constitutive components of secretion were assessed after 2-h incubations with 5 mM 8-Br-cAMP or control. cAMP treatment led to a 2-fold increase in hGH secretion relative to control. Cotransfection of wild-type Dexras1 had no effect on cAMP-stimulated hGH secretion, but a constitutively active mutant, Dexras[A178V], attenuated stimulated secretion by 86% (P < 0.01). A double-mutant containing a deletion of the carboxyl terminus isoprenylation site, Dexras[A178V/C277term], did not inhibit cAMP-stimulated hGH secretion, indicating that the effect is prenylation dependent. These findings suggest that activation of Dexras1 has important functional consequences leading to inhibition of stimulus-secretion coupling in corticotroph cells. Because Dexras1 messenger RNA is strongly and rapidly induced during glucocorticoid treatment, these results raise the possibility that Dexras1 may participate in the signal transduction pathways that govern the rapid regulatory effects of glucocorticoids on peptide hormone secretion in corticotroph cells.