Assessment of adrenal reserve and the diagnosis of adrenal insufficiency by acute adrenocortical stimulation with ACTH-(1–24) has been well established. Alternatively, estimation of adrenocortical enzymatic activities by this method for the detection of inherited or acquired biosynthetic abnormalities has been less well characterized. Some of the discrepancies between studies estimating adrenocortical enzymatic activities in different pathological conditions (e.g. hyperandrogenism) may result from the different stimulation protocols used. The objective of this prospective study was to establish the inherent variability of the adrenal response to acute ACTH-(1–24) stimulation and to determine the effect of sampling time, stimulation dose, and subject weight on the same. Forty-one normal female volunteers were recruited (mean age, 29.1 yr), 30 within 90–110% ideal body weight and 11 weighing more than 120% ideal body weight. Three protocols were designed to study 1) the effects of sampling time, ACTH-(1–24) dose, and subject weight on adrenal response; 2) the effect of time of the day on the variability of basal steroid levels and the adrenal response to stimulation; and 3) the long term reproducibility of the adrenal response to ACTH-(1–24). Androstenedione, 17-hydroxyprogesterone, 11-deoxycortisol, dehydroepiandrosterone, and cortisol were measured in serum under basal and stimulated conditions. All subjects had normal basal levels of testosterone, androstene-dione, dehydroepiandrosterone sulfate, and PRL. The acute iv administration of 0.10, 0.25, and 1.0 mg ACTH-(l–24) elicited similar and maximal steroid responses, with all steroid levels reaching a plateau 60–90 min poststimulation regardless of subject weight. Sampling of basal steroid levels every 5 min in the morning (AM; beginning 0700–0900 h) or evening (PM; 1500–1700 h) did not reveal any difference in steroid variability. Only the mean basal cortisol level was higher in AM than PM testing (P < 0.03). Although the mean levels of dehydroepian-drosterone and 17-hydroxyprogesterone 60 min after stimulation were significantly higher in AM than PM studies, these differences were minimal. Ten volunteers underwent an average of four (range, 2–6) adrenal stimulation studies using 1.0 mg ACTH-(l-24) over a 1-yr period. The long term coefficient of variation (CV) for basal steroid levels ranged from 15-28%. Calculations of net adrenal response (Δ steroido-t and area Δ steroido-t) were less reproducible (CV, 0–82%) than measures of absolute response (steroidt, area steroidt, and %steroidt; CV, 7–32%). This difference in CV between the measures of net and absolute adrenal responses was significant for all steroids except androstenedione. No significant difference (P > 0.05) in adrenal response was observed for studies in the same individual performed on either days 3–5 or 6–8 of the menstrual cycle. The intrinsic variability of the adrenal response to acute ACTH-(1–24) stimulation must be taken into account when attempting to establish adrenocortical biosynthetic differences in the same population. Furthermore, studies using acute ACTH-(l-24) stimulation for the determination of enzymatic activities using stimulatory doses ranging from 0.1–1.0 mg or performed either in the AM or PM are comparable. © 1990 by The Endocrine Society.