Certain classes of prohormones and other neuroendocrine or endocrine-derived secretory proteins are post-translationally modified in the secretory storage granules. If such molecules were to be biosynthesized to acceptable quantity and yield using endocrine-derived cell lines, it would be important to understand the relationship between the secretory dynamics and the conversion and release of the immature and mature forms of the molecule. We studied aspects of such a relationship using the endocrine-derived cell line โคTC-3, which synthesizes murine proinsulin, sequesters it into secretory granules, and converts it into mature insulin. In T-flask experiments with confluent cultures of โคTC-3 cells, intracellular and secreted (pro)insulin was sampled before and after episodes of stimulated exocytosis and recharging and quantified by radioimmunoassay and reversedphase high-performance liquid chromatography (HPLC). Under conditions of steady-state secretion in glucoserich growth medium the cells turned over their (pro)insulin inventory (90 ยฑ 5% mature insulin) at 2-3% per hour through secretion of (pro)insulin which was less than 70% mature. During an episode of hyperstimulated exocytosis induced by the combined secretagogues carbachol (1 ยตM) and isobutylmethylxanthine (1 mM), โผ80% of the intracellular (pro)insulin stores were depleted within 2 h and 84 ยฑ 4% of the secreted (pro)insulin was in the mature form. Following the discharging episode, exocytosis was suppressed to 10% of its steady-state rate with a treatment which attenuated calcium influx (20 ยตM verapamil with reduced levels of calcium in the medium). Under this condition the secreted protein was only โผ50% converted to mature insulin, but 85 ยฑ 10% of the net (pro)insulin accumulating within the intracellular stores was converted to the mature form. The inverse relationship between rate of secretion and degree of conversion of secreted (pro)insulin is consistent with a previously observed phenomenon of preferential basal secretion from immature secretory granules. This tends to enrich the secreted peptides in immature forms relative to the total intracellular pool. Preferential early secretion can best be overcome by rapid discharging of the long-term and predominantly mature stores. Thus, a cyclic con-trolled secretion process wherein product is collected during intermittent discharging episodes would provide a better yield of mature product than would steady-state secretion.