The ACTH-adrenal axis is a critical stress-responsive system with prominent circadian rhythmicity. To test the basis for the circadian ACTH physiology, we have used 1) a sensitive and specific two-site immunoradiometric assay to estimate plasma ACTH-(1-39) concentrations during intensive (every 10 min) and extended (24-h) blood sampling to capture complete diurnal ACTH profiles in eight normal men, and 2) a novel deconvolution model designed to resolve the number, amplitude, and duration of ACTH secretory bursts and simultaneously estimate subject-specific ACTH half-lives under physiological conditions in vivo. Deconvolution revealed 40 +/- 1.5 significant ACTH secretory bursts/24 h, with a mean interburst interval of 39 +/- 2.3 min. ACTH secretory bursts were discrete punctuated events arising without tonic interpulse secretion and had a half-duration (duration at half-maximal amplitude) of 19 +/- 2 min. The estimated half-life of endogenous ACTH was 15 +/- 1.2 min, and its daily production rate was 0.96 +/- 0.16 ng/mL (0.21 +/- 0.035 nmol/L) distribution volume. Cosinor analysis revealed a significant (3.8-fold) 24-h rhythm in the mass (or rate) of ACTH secreted per burst (maximal at 0818 h), but no nyctohemeral variation in ACTH secretory pulse frequency. The validity of ACTH pulse analysis was supported by the significantly nonrandom associations among ACTH, beta-endorphin, and cortisol peaks in the same subjects. Specifically, we found that ACTH and beta-endorphin bursts occurred simultaneously (P less than 10(-4)) and were both followed in 10 min by a cortisol pulse (P less than 10(-4)). We conclude that 1) selective amplitude control of a punctuated burst-like mode of ACTH secretion can give rise to the nyctohemeral corticotropic rhythm without the need to postulate any tonic (or interpulse basal) component of ACTH release; and 2) there is exquisite 3-fold temporal synchrony among bursts of ACTH, beta-endorphin, and cortisol release in normal men.