In the dog's saphenous vein acetylcholine inhibits the norepinephrine release caused by nerve stimulation, but not that caused by tyramine. Experiments were performed to determine whether acetylcholine affects the release of norepinephrine evoked by high K+ concentrations. We recorded changes in isometric tension of dog saphenous vein strips. Acetylcholine (5 X 10(-9) to 10(-6) g/ml) caused dose-dependent relaxations during contractions caused by K+ = 40 mEq/liter. These relaxations were not depressed by tetrodotoxin (10(-7) g/ml), which abolished the response to nerve stimulation, but were inhibited by atropine (10(-7) g/ml). Strips of saphenous veins were incubated with [3H]norepinephrine and mounted for superfusion (3 ml/min) and isometric tension recording; the total radioactivity and the amount of intact [3H]norepinephrine present in the superfusate were determined. K+ at 50 mEq/liter increased tension, total radioactivity of the superfusate, and the [3H]norepinephrine afflux; acetylocholine (10(-7) g/ml) depressed the contractions and diminished the efflux of [3H]norepinephrine. Increasing the K+ concentration from 50 to 70 mEq/liter augmented both tension and the evoked release of [3H]norepinephrine. Acetylcholine did not significantly alter the release of [3H]norepinephrine evoked by K+ = 120mEq/liter. These experiments show that acetylcholine inhibits the norepinephrine release evoked by potassium ions, as it does during nerve stimulation. The inhibition of adrenergic neurotransmission is not due to interference with action potential electrogenesis, but probably is caused by hyperpolarization of the adrenergic nerve endings.