The investigation reported here was designed to gain further insight into the mechanisms by which phenylethanolamine N-methyltransferase (PNMT) is regulated. Explants of adrenal medullae were cultured in defined media for up to 48 h, during which time the tissue remained histologically intact. Addition of epinephrine to the medium led to a diminution in the activity of PNMT, measurable in the dialyzed homogenates of the cultured tissue. The enzyme activity was inversely proportional to the concentration of epinephrine present in the culture medium. A diminution in the amount of PNMT protein also resulted from incubation of the explants in the presence of epinephrine. The extent of loss of PNMT, measured by immunochemical titration, corresponded to the degree of loss in PNMT activity under the same conditions. None of the metabolites of epinephrine, including 3,4-dihydroxy- or 3-methoxy-4-hydroxymandelate, or metanephrine, had an effect on PNMT. Tyrosine hydroxylase and catechol O-methyltransferase activities were also diminished, whereas tyrosine transaminase, acid phosphatase, and monoamine oxidase activities were not affected by addition of epinephrine to the medium. Ascorbic acid added to the medium prolonged the lifetime of epinephrine but did not alter the degree of diminution of PNMT. The results obtained are consistent with the view that epinephrine is rapidly assimilated into the cytoplasm of medullary cells and plays an important role in regulating the concentration of PNMT in the adrenal gland.