3-Methoxy-4-hydroxyphenylglycol (MHPG) is formed by the sequential actions of monoamine oxidase (MAO) and catechol-O-methyltransferase on norepinephrine within extraneuronal tissues or by extraneuronal O-methylation of 3,4-dihydroxyphenylglycol (DHPG) produced intraneuronally from norepinephrine. This study examined the contributions of neuronal and extraneuronal norepinephrine metabolism to formation of MHPG in rats. Spillover of MHPG into plasma (605 +/- 28 pmol/kg per min) was higher than spillover of DHPG (463 +/- 15 pmol/kg per min), norepinephrine (165 +/- 9 pmol/kg per min) and normetanephrine (56 +/- 4 pmol/kg per min). Comparison of MHPG spillover with the increment in normetanephrine spillover after MAO inhibition (81 pmol/kg per min) combined with the spillover of MHPG derived from circulating normetanephrine (21 pmol/kg per min) indicated that only 17% of plasma MHPG is derived from the extraneuronal deamination and O-methylation of norepinephrine; the remaining 83% is from DHPG produced by deamination of norepinephrine within neurons. The production of MHPG from infused DHPG indicated that 30% of the MHPG in plasma is produced by O-methylation of DHPG after entry of DHPG into the bloodstream and 53% (83-30) from metabolism of DHPG before its entry into the bloodstream. Metabolism of circulating norepinephrine made a minor (1-3%) contribution to plasma concentrations of DHPG and MHPG, but a much larger (46%) contribution to plasma normetanephrine. The results provide a comprehensive examination of norepinephrine metabolism by O-methylation and deamination pathways. Since MHPG is the principal norepinephrine metabolite excreted in the rat, the findings also show that total body turnover of norepinephrine is dependent mainly on neuronal metabolism of the transmitter; at rest extraneuronal pathways contribute as little as 15% to norepinephrine turnover.