Cells prepared from a transplantable rat pheochromocytoma synthesize norepinephrine from 14C-tyrosine, at a rate of 9.4 +/- 0.5 pml/min/mg of protein, in vitro. Incubation of the cells in a medium containing 56 mM K+ results in a 2- to 6-fold increase in norepinephrine synthesis. This increase in norepinephrine synthesis is dependent upon the presence of Ca++ in the incubation medium. Stimulation of the cells by 56 mM K+ increases the conversion of tyrosine to dopa in the presence of brocresine (an inhibitor of aromatic L-amino acid decarboxylase), and has no effect on the conversion of 3H-dopa to norepinephrine. Cells can be depleted of up to 70% of their catecholamine stores by prior incubation in 56 mM K+. Norepinephrine synthesis in catecholamine-depleted cells incubated under control conditions in only slightly (20-40%) greater than it is in nondepleted cells. However, 56 mM K+ PRODUCES A SIMILAR INCREASE IN NOREPINEPHRINE SYNTHESIS IN DEPLETED CELLS AS IT DOES IN NONDEPLETED CELLS. Inhibition of amine oxidase (flavin containing) by preincubaiton with pargyline does not greatly affect catecholamine synthesis. Incubation of the cells in 56 mMK+ results in an increase in tyrosine 3-monooxygenase activity. These results indicate that the depletion of catecholamine stores plays only a minor role in the increase in norepinephrine synthesis caused by the stimulation of chromaffin cells and suggest that the activation of tyrosine 3-monooxygenase plays a more important role in this phenomenon.