The presence and secretion of immunoreactive dynorphin in bovine adrenal medulla and isolated adrenal chromaffin cells were examined and compared with those of immunoreactive leucine-enkephalin. Using an antiserum raised against dynorphin-(1-13), a sensitive and highly specific radioimmunoassay was developed. A nearly intact antigen was required for recognition by the antiserum since it did not react with leucine-enkephalin and reacted poorly with dynorphin-(1-12) (cross-reactivity 0.5%) and other fragments of dynorphin-(1-13). On the other hand, the antibody used for detection of leucine-enkephalin did not cross-react with dynorphin(1-13). Adrenal medulla acid extracts contained 195 times more immunoreactive leucine-enkephalin than dynorphin. However, the concentration of immunoreactive dynorphin in acid extract of freshly isolated adrenal chromaffin cells was only 1.4 times smaller than that of leucine-enkephalin. Incubation of the isolated chromaffin cells in the presence of acetylcholine, nicotine, high potassium, but not muscarine, induced a concomitant release of immunoreactive dynorphin (3.5-9% of total cell content) and leucine-enkephalin (6.5-11.4% of total cell content).