Abstract: This paper reports a reversed-phase high-performance liquid chromatography with electrochemical detection to separate and determine monoamines and their metabolites (norepinephrine, epinephrine, dopamine, 5-hydroxytryptamine, 3,4-dihydroxyphenylglycol, 3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenyacetic acid, homovanillic acid and 5-hydroxyindole-3-acetic acid) simultaneouslyl in rat brain, using sodium octyl sulfate and dibutylamine as counter-ions, and isoproterenol as internal standard. The supernatants of protein-precipitated rat brain tissue homogenates were directly injected into the chromatographic system. The chromatographic conditions such as mobile phase pH, concentration of methanol, counter-ions, and applied potential of working electrode, etc., were discussed. The monoamines and their metabolites in rat hypothalamus were determined.