In the isolated rat vas deferens the release and intraneuronal disposition of endogenous norepinephrine (NE) were compared with those of newly synthesized or exogenous radioactive NE by preloading tissues with trace amounts of tritiated dopamine ([3H]DA) or tritiated NE ([3H]NE) and measuring release of radioactive and endogenous NE and dihydroxyphenylglycol (DHPG). Tissues were examined before and during electrical simulation, exposure to tyramine or exposure to depolarizing concentrations of K+. In [3H]DA-preloaded tissues the [3H]DA was converted readily to [3H]NE. Newly synthesized radioactive NE formed from exogenous [3H]DA was distributed differently from endogenous NE within at least two intraneuronal pools. One pool contained a high concentration (high specific activity) of newly synthesized [3H]NE and less than 3% of the total NE content of the tissues, and it released NE more readily than the larger low specific activity pool which contained over 95% of the total tissue NE content. Exogenous [3H]NE in [3H]NE-preloaded vasa deferentia was distributed among at least three different tissue pools, one consisting of extraneuronally bound NE containing [3H]NE of high specific activity, and two intraneuronal pools containing [3H]NE of intermediate and low specific activity in which NE was released from the intermediate specific activity pool more readily than from the low specific activity pool.(ABSTRACT TRUNCATED AT 250 WORDS)