Differential secretion of norepinephrine and epinephrine was studied in cultured bovine chromaffin cells. Nicotinic agonists and 55 mM K+ evoked a slightly greater release of norepinephrine than of epinephrine: The percentage of norepinephrine secreted was 1.5 to two times greater than the percentage of epinephrine secreted. In contrast, when the cells were treated with phorbol 12,13-dibutyrate, the percentage of norepinephrine released was six to eight times greater than that of epinephrine released. Similar results were obtained in experiments with cultures highly enriched in either norepinephrine-containing cells or epinephrine-containing cells. In response to 55 mM K+, catecholamine release from norepinephrine-containing cells was two times greater than that from epinephrine-containing cells. In response to phorbol 12,13-dibutyrate, secretion from norepinephrine-containing cells was 13 times greater than that from epinephrine-containing cells. These results suggest that protein kinase C plays a specific role in the regulation of catecholamine secretion from norepinephrine-containing cells.