Chromaffin cells in primary culture take up [14C]ascorbic acid from the incubation medium. Cells, stimulated immediately after a short labeling period with [14C]ascorbate, secrete ascorbic acid concomitantly with catecholamines (CA) through a nicotinic receptor-mediated Ca2+-dependent process. A proportional release of CA and [14C]ascorbic acid was observed through a large range of secretion rates obtained by varying the concentration of nicotine or by changing the concentrations of Ca2+ and Na+ in the external medium. However, under the same conditions of stimulation, different cell preparations secrete 2-10 times more CA than [14C]ascorbate (as percentage of cell content). Furthermore, a different time course of secretion was observed for CA and [14C]ascorbate for each of several secretagogues. In addition, Ba2+ is a much more potent stimulus for CA secretion than for secretion of [14C]ascorbate, and Ca2+ channel blockers are more potent in inhibiting CA secretion than [14C]ascorbate secretion. These data suggested the possibility that newly taken up ascorbate was being secreted from a compartment altogether distinct from the chromaffin vesicle. This hypothesis was confirmed by subcellular distribution studies, where only a minor fraction of newly taken up [14C]ascorbate was found in the vesicular fraction (P2) from homogenates of chromaffin cells prepared after a short incubation with [14C]ascorbate. However, the subcellular distribution of [14C]ascorbate follows that of endogenous ascorbate when a short pulse with the label is chased by a prolonged equilibration period in the absence of ascorbate, indicating that a transfer has occurred from the extravesicular compartment(s) to the CA storage organelle. Endogenous ascorbate, which is found both inside and outside the chromaffin vesicle, was also found to be secreted from chromaffin cells, indicating that ascorbic acid could be released simultaneously from two different subcellular compartments.