Objective: To determine the sensitivity of 11β-hydroxyandrostenedione (11-OHA4) and Δ5-androstenediol (ADIOL) as markers of excessive adrenal androgen production. Design: Prospective study. Setting: Academic medical centers. Patient(s): Thirteen women with untreated 21-hydroxylase-deficient nonclassic adrenal hyperplasia (NCAH) and 18 healthy, eumenorrheic, nonhirsute controls matched for age and body mass index. Intervention(s): All subjects were studied before and after acute adrenal stimulation with 0.25 mg of IV ACTH-(1-24). Main Outcome Measure(s): Basal levels of total testosterone, sex hormone-binding globulin, DHEAS, and free testosterone were measured. Levels of androstenedione (A4), DHEA, 11-OHA4, and ADIOL were determined before (Steroid0) and 60 minutes after (Steroid60) acute ACTH- (1-24) stimulation. Result(s): Patients with NCAH had higher median basal levels of DHEAS and total and free testosterone than controls. Patients with NCAH had higher median A40, A460, DHEA0, DHEA60, 11-OHA40, ADIOL0, and ADIOL60 levels but similar 11-OHA460 levels compared with controls. Among patients with NCAH, 30%, 54%, 15%, and 85% had 11-OHA40, ADIOL0, 11- OHA460, and ADIOL60 levels, respectively, above the 95th percentile of controls. Conclusion(s): Overall, serum levels of 11-OHA4 did not appear to be a very sensitive marker of excessive adrenal androgen production, at least in patients with NCAH. Although ACTH-stimulated ADIOL levels were elevated in 85% of the patients studied, they did not appear to have any advantage over the measurement of A4 or DHEA levels.