The importance of the process of continuous biosynthesis of locust adipokinetic hormones (AKHs) for the availability of these peptide hormones for release was assessed in vitro by inhibiting this biosynthesis followed by secretory stimulation. Inhibition of the biosynthetic activity for AKHs by brefeldin A caused a considerable inhibition of the AKH release induced by the endogenous crustacean cardioactive peptide (CCAP). After brefeldin A treatment followed by potassium depolarization, CCAP-induced AKH release was completely abolished. In vitro pulse-chase labeling experiments indicated that constitutive secretion from the AKH-producing cells does not occur. It is concluded that AKH secretion involves a regulated release from a relatively small pool of newly formed secretory granules, while older AKH-containing granules appear to be unavailable for release.