To elucidate the origin and significance of dihydroxyphenylglycol (DHPG) as a metabolite of norepinephrine (NE), the isolated rat vas deferens was preloaded with tracer amounts of tritiated NE and examined for the release of radioactive and endogenous NE and DHPG before and during electrical stimulation or stimulation with excess K+. Tissues were incubated with desipramine or reserpine to determine the effects of blockade of neuronal uptake and of interference with vesicular translocation of NE. Radioactive NE appeared to distribute differently from endogenous NE into at least two pools, but for the most part endogenous NE and DHPG behaved similarly in response to pharmacological manipulations. Desipramine blocked completely the increased appearance of both radioactive and endogenous DHPG in the medium during electrical stimulation or K+ stimulation; DHPG responses to stimulation are thus dependent on recapture of NE at the synapse. Basal release of DHPG was increased by reserpine, and this increase was not affected by desipramine; therefore, reserpine-induced release of DHPG is independent of neuronal uptake consistent with formation of DHPG from NE leaking into the cytosol from vesicular stores. Reserpine enhanced the release of DHPG during stimulation, and concomitant desipramine treatment blocked this effect; thus, interference with NE translocation into storage vesicles increases the availability of recaptured NE for intraneuronal metabolism. During stimulation of NE release between 70 to 80% of the recaptured NE was estimated to be sequestered into storage vesicles for rerelease. Combined measurement of endogenous and labeled NE and DHPG provides a useful tool for examining neuronal uptake and intraneuronal disposition of NE.