The release of newly synthesized neuropeptides was studied in an in vitro system using the adipokinetic hormone (AKH)-producing cells of an insect (Locusta migratoria) as a model system. Tritiated phenylalanine incorporated into three hormonal neuropeptides, AKH I, II and III, was used to distinguish newly synthesized hormones from older, preexisting ones. After pulse-chase labeling experiments of varying duration, the secretion of AKHs by the AKH cells was stimulated. Both hormones released into the incubation medium after stimulation and non-released hormones extracted from the tissue were separated by reversed-phase high performance liquid chromatography. Their radioactivity was measured by scintillation counting of the column eluate. The ratio between the specific radioactivities of the released and the non-released neuropeptides was always greater than 1.0, which indicates that the newly synthesized peptides are preferentially released. The percentages of newly synthesized (radioactive) AKHs which are released, increased until 8 1/4 h and decreased thereafter. The results indicate that after the packaging of the prohormones into secretory granules and their processing to bioactive AKHs, some further maturation of the secretory granules is required before they can release their content. After an 8 1/4 h incubation, secretory granules with radioactive AHKs enter a non-releasable pool consisting of older secretory granules.