The current study examines the effects of amitriptyline administration on extracellular levels of monoamines and their metabolites in hypothalamus of conscious rat using in vivo microdialysis. Systemic amitriptyline (30mg/kg, IP) maximally increased extracellular norepinephrine to nearly 19 times, dopamine 12 times, and serotonin 2.5 times that of their respective basal values. Local administration of amitriptyline (10μM) in the perfused artificial cerebrospinal fluid increased extracellular serotonin levels to 2.7 times basal levels - a similar increase to that observed with systemic administration. In contrast, local administration of amitriptyline (10μM) increased extracellular norepinephrine and dopamine to only 5.3 and 3.8 times basal levels. Systemic administration of amitriptyline (30 mg/kg, IP) during local amitriptyline perfusion increased norepinephrine and dopamine levels to 28 and 12.9 times their respective basal levels without affecting serotonin levels. Systemic amitriptyline administration preferentially increases extracellular norepinephrine and dopamine levels over serotonin levels in the rat hypothalamus and the effects on the catecholamine levels may be partially due to mechanisms other than local uptake inhibition.