The overflow of endogenous norepinephrine caused by transmural electrical stimulation or depolarization with potassium was smaller in superfused segments of the rabbit carotid artery with intact endothelium than in segments denuded of endothelium. In segments preincubated with [3H]norepinephrine, the lesser overflow was found to be partially due to metabolism by the endothelium of the neurotransmitter. Even after treatment to block the disposition of norepinephrine, the endothelium acted as a partial physical barrier to the overflow of norepinephrine into the lumen of arteries superfused and perfused selectively. However, a lesser overflow of norepinephrine to the adventitia of the artery accounted for the majority of the difference in overflow between segments with and without endothelium. The inhibition by the endothelium of the overflow of norepinephrine from adrenergic nerves was unaffected by blocking prejunctional alpha 2-adrenoceptors, prostaglandin synthesis, free radicals, or guanylate cyclase. Vasodilators released from the endothelium of a donor artery inhibited contractions caused by adrenergic nerve stimulation of a bioassay artery but failed to inhibit norepinephrine release. These observations indicate that the endothelium 1) metabolizes norepinephrine, 2) acts as a physical barrier to its overflow into the blood vessel lumen, and 3) inhibits the release of the adrenergic transmitter from adrenergic nerves.