The overflows of 3,4‐dihydroxyphenylalanine, dopamine, noradrenaline, and 3,4‐dihydroxyphenylglycol in canine portal vein superfused in vitro were studied before, during, and after depolarization of sympathetic nerve endings. The four compounds were separated from superfusate and from tissue on Sep‐Pak C‐18 cartridges and quantified by HPLC with electrochemical detection. Physiological and biochemical methods were used to show that the compound released was most probably 3,4‐dihydroxyphenylalanine; the identity of the other endogenous compounds has been established previously. Release of 3,4‐dihydroxyphenylalanine was calcium and frequency dependent, inhibited by α‐m‐L‐p‐tyrosine (an inhibitor of tyrosine hydroxylase) and augmented by 3‐hydroxybenzylhydrazine (an inhibitor of aromatic amino acid decarboxylase). The overflows of dopamine, noradrenaline, and 3,4‐dihydroxy phenylglycol from the vein were calcium and frequency dependent. It was estimated that under control conditions, ∼ 80% of the total 3,4‐dihydroxyphenylalanine that was synthesized was directed to catecholamine biosynthesis, ∼8% overflowed from the vein, and ∼ 14% remained unchanged within the tissue. It is concluded that 3,4‐dihydroxyphenylalanine and dopamine are released together with noradrenaline and 3,4‐dihydroxyphenylglycol from portal vein upon nerve depolarization.