Enkephalins, vasoactive intestinal polypeptide, and chromogranin A are all contained in the secretory vesicles of chromaffin cells in culture, and are all released from this compartment by secretagogues in a calcium-dependent way. The biosynthesis of each of these peptides, however, is under quite independent regulation. The synthesis and secretion of enkephalin is tightly coupled to acetylcholine and elevated potassium stimulation by calcium influx. Once calcium enters the cell, calcium acts at pharmacologically distinct sites to elicit secretion and enhanced biosynthesis of Metenkephalin. This is demonstrated by the calcium-independent stimulation of enkephalin secretion by 1 mM barium, in contrast to the dependence on extracellular calcium of barium-stimulated biosynthesis of this peptide. The synthesis and secretion of VIP is also coupled to acetylcholine and elevated potassium stimulation by calcium influx. Treatment with barium demonstrates that calcium acts at distinct sites to stimulate secretion and biosynthesis of this peptide; however induction of VIP by barium and veratridine shows greater sensitivity to the calcium channel blocker methoxyverapamil (D600) than does the induction of Met-enkephalin by these agents. These differences in D600 sensitivity may be due to differences in calcium metabolism or voltage-dependent calcium channels in enkephalin-producing and VIP-inducible subpopulations of chromaffin cells. Chromogranin A levels are essentially unaffected by any of the agents which increase enkephalin and VIP levels, although it is secreted in parallel with enkephalins and catecholamines from chromaffin cells in response to secretagogues. We suggest that peptide hormones such as VIP and enkephalins are regulated by calcium-dependent stimulus-secretion-synthesis coupling in the chromaffin cell. Cyclic AMP is a positive regulator of enkephalin and VIP biosynthesis, but does not affect acute release of these peptides. The cAMP/protein kinase A system may be a distal mediator of peptide biosynthesis stimulated by secretagogues. Alternatively, cAMP may be involved in early developmental establishment of phenotype or long-term regulation of peptide biosynthesis by other hormones or neurotransmitters. Chromogranin A may represent a class of intravesicular, soluble proteins that are expressed constitutively by the chromaffin cell in the presence or absence of positive regulators of other systems. The biosynthesis of chromogranin A may be coupled to the production or assembly of the secretory vesicle itself.