In canine blood vessels acetylcholine and adenosine inhibit the exocytotic release of norepinephrine during nerve stimulation. The present experiments were designed to determine the Ca2+ dependence of these prejunctional effects. Segments of canine saphenous veins were mounted for isometric tension recording in organ chambers filled with Krebs-Ringer or Tyrode solution. Altering the Ca2+ concentration of the solution did not affect the inhibitory response to acetylcholine during nerve stimulation; the prejunctional potency of adenosine was inversely related to the Ca2+ concentration of the bath content. the ionophore A23187 caused contractions which were inhibited by phentolamine, verapamil, and adenosine but were augmented by acetylcholine. Helical strips of dog saphenous veins were incubated in [3H]norepinephrine and mounted for superfusion and determination of [3H]norepinephrine in the superfusate. A23187 increased the overflow of [3H]norepinephrine. Acetylcholine augmented this efflux; by contrast adenosine decreased the release induced by the ionophore. The results demonstrate that the prejunctional effect of acetylcholine was not due to direct interference with the availability of Ca2+ for the electro-secretory process in adrenergic nerve terminals and suggest that adenosine interferes either with the coupling role of the activator ion or its extrusion from the neuroplasm.