A rapid and sensitive set-up consisting of a microbore HPLC with electrochemical detection coupled with two serial on-line microdialysis systems was described to simultaneously measure endogenous levels of acetylcholine (ACh, choline (Ch), dopamine (DA); 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in freely moving rats. The validity of the present procedure is demonstrated by analyzing the effect of methamphetamine (2 mg/kg, s.c.) on the increase release of DA, 3-MT, 5-HT, and ACh but without effect in Ch, DOPAC, HVA and 5-HIAA from the striatum of freely moving rat.