Glucocorticoid is reported to regulate catecholamine synthesis and storage. However, it is not clear whether the actual amount of catecholamine released from individual granules (quantal size, Q) in mature chromaffin cells is affected by glucocorticoid. Using carbon fiber amperometry, we found that dexamethasone did not affect the mean cellular Q or the proportional release from the different populations of granules in rat chromaffin cells cultured for 1 day in a serum-free, defined medium. After 2 extra days of culture in the defined medium, there was a rundown in mean cellular Q and it was associated with a shift in the proportional release from the different granule populations. This phenomenon could not be rescued by serum supplement but could be prevented by dexamethasone via an action that was independent of changes in voltage-gated Ca²⁺ channel (VGCC) density. Using simultaneous measurements of membrane capacitance and cytosolic Ca²⁺ concentration ([Ca²⁺]_i), we found that for cells cultured in defined medium, dexamethasone enhanced the exocytotic response triggered by a brief depolarization (50 ms) without affecting VGCC density or the fast exocytotic response triggered via flash photolysis of caged Ca²⁺. Thus, glucocorticoid may regulate the number of immediately releasable granules that are in close proximity to a subset of VGCC. Since chromaffin cells in vivo are exposed to high concentrations of glucocorticoid, our findings suggest that the paracrine actions of glucocorticoid maintain the mean catecholamine content in chromaffin cell granules as well as the co-localization of releasable granules with VGCC.