A direct method has been employed to estimate the rate of production by human brain of 3-methoxy-4-hydroxyphenethyleneglycol, the major metabolite of brain norepinephrine, a brain neurotransmitter. Venous specimens were obtained from the internal jugular vein from ten awake human subjects at a puncture site above the common facial vein, the first major source of extracranial inflow. Arterial specimens were simultaneously obtained from the radial artery. Plasma samples were assayed and a highly significant difference was found in the concentration of the metabolite in plasma coming out of the brain (venous blood) as compared to plasma entering the brain (arterial blood). This venous-arterial difference was calculated to be 0.7 +/- 0.1 nanogram per milliliter of blood. Assuming an adult brain weight of 1400 grams and normal cerebral blood flow, it is estimated that the rate of production of 3-methoxy-4-hydroxyphenethyleneglycol by the awake human brain is approximately 597 nanograms per minute or 35.8 micrograms per hour. Urine specimens were also collected from six of these subjects during a period of 1 to 3.5 hours, which bracketed the time the blood samples were obtained. For these six subjects the output of 3-methyoxy-4-hydroxyphenethyleneglycol by whole brain was estimated to be 40.9 micrograms per hour, whereas the rate of its excretion into urine was 64.5 micrograms per hour.