The distal convoluted tubule (DCT) reabsorbs ~7% of the filtered sodium load and contributes importantly to blood pressure regulation. The Na-Cl co-transporter (NCC) is the major route for apical Na entry into DCT cells. Thiazide diuretics inhibit this NCC and are a mainstay hypertension treatment. Previous studies have shown that both mineralocorticoids and glucocorticoids can increase NCC expression. Although glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) are expressed throughout the distal nephron, this part of the renal tubule is conventionally thought to be aldosterone-sensitive due to 11b-hydroxysteroid dehydrogenase (11bHSD2) coexpression. However, we find that 11bHSD2, which inactivates glucocorticoids, seldom colocalises with NCC, suggesting that glucocorticoids may directly activate NCC. We therefore investigated whether glucocorticoids activate NCC in vivo and whether GR or MR is involved in this process. Male C57BL6 mice were anaesthetized under isofluorane and implanted (s.c.) with silastic pellets containing spironolactone (50 mg, chronic MR blockade) or RU486 (60 mg, chronic GR blockade). Control mice received no implant. After 5 days, corticosterone (cort, 6 mg/kg, s.c.) or vehicle (veh, 2% DMSO, s.c.) was injected at ~8am, coincident with the nadir of the endogenous corticosterone circadian rhythm. 4 hours later mice were culled and kidneys snap frozen. NCC threonine phosphorylation (on T53, pT53-NCC) was measured by western analysis as an index of transporter activation. Data are mean±SD. In control mice, acute cort doubled pT53-NCC abundance (1.0±0.49 AU (veh) vs 1.9±0.28 AU (cort), p=0.004, Student’s t test, n=6 in each group). Chronic MR blockade reduced total NCC protein (0.39±0.21 AU (spironolactone; n=5) vs 0.80±0.18 AU (control; n=6), p=0.004, by post hoc Tukey tests) but acute cort still substantially increased pT53-NCC abundance (1.0±0.46 AU (veh; n=5) vs 3.7±1.4 AU (cort; n=7), p=0.0018, Student’s t test). In contrast, chronic GR blockade did not affect total NCC abundance (0.59±0.15 AU (RU486) vs 0.80±0.18 AU (control) p=0.15, Student’s t test, n=6 in each group) but did prevent phosphorylation induced by acute cort injection (1.0±0.19 AU (veh) vs 1.0±0.15 (cort), p=0.51, Student’s t-test n=6 in each group). These data suggest MR activation is required for maintenance of tonic levels of NCC protein. They further show that glucocorticoids can regulate NCC function by acutely increasing NCC phosphorylation through GR activation. This enhanced NCC activity has implications for blood pressure control, particularly in individuals with inappropriately elevated glucocorticoids, such as in Cushing’s syndrome, metabolic syndrome, or during chronic stress.