Existing glucocorticoid treatment for congenital adrenal hyperplasia (CAH) is suboptimal and nonphysiological. We compared hormonal profiles during therapy with a new modified-release hydrocortisone (MR-HC), Chronocort, to conventional hydrocortisone (HC), Cortef, in patients with CAH. We conducted a Phase 2, open-label, crossover pharmacokinetic and pharmacodynamic study in 14 patients (out of whom seven were male subjects, age ranging from 17 to 55) with classic 21-hydroxylase deficiency. One week of thrice daily HC (10, 5 and 15 mg) was followed by 1 month of once daily MR-HC (30 mg at 22:00 hours). Twenty four-hour sampling of cortisol, 17-hydroxyprogesterone (17-OHP), androstenedione, and ACTH was performed at steady state. The primary outcome measures were 8- and 24-h area under the curve (AUC) hormones and 08:00 hours 17-OHP. Hydrocortisone therapy resulted in three cortisol peaks. A single cortisol peak occurred at approximately 06:00 hours on MR-HC. MR-HC resulted in significantly (P < 0.001) lower 24-h afternoon (12:00 to 20:00 hours), and night-time (20:00 to 04:00 hours) cortisol as compared with HC. From 04:00 to 12:00 hours, when physiological cortisol is highest, cortisol was higher on MR-HC than HC (P < 0.001). Patients on MR-HC had significantly (P < 0.05) higher afternoon (12:00 to 20:00 hours) 17-OHP, androstenedione and ACTH, but significantly (P = 0.025) lower 08:00 hours 17-OHP. No serious adverse events occurred. Modified-release hydrocortisone represents a promising new treatment for CAH. Overnight adrenal androgens were well-controlled, but rose in the afternoon with once-daily dosing suggesting that a morning dose of glucocorticoid is needed. Further studies are needed to determine the optimal dosing regimen and long-term clinical outcome.