Two distinct norepinephrine (NE) transporter mechanisms (uptake 1 and uptake 2) regulate extracellular NE concentrations. An association has been observed between the gradual improvement in patients treated with antidepressants that inhibit the NE transporter (NET/uptake 1) and increases in urinary normetanephrine, the O-methylated NE metabolite and potent inhibitor of uptake 2. These observations led to the hypothesis that increased levels of normetanephrine, and consequently inhibition of uptake 2, may partly be responsible for the clinical efficacy of some antidepressants. To investigate this hypothesis, we employed microdialysis techniques in the rat frontal cortex to monitor extracellular changes in normetanephrine following chronic administration of the clinically effective antidepressant, venlafaxine (a serotonin (5-HT) and NE reuptake inhibitor). We evaluated the neurochemical effects of inhibiting uptake 2 alone, or in conjunction with venlafaxine, on extracellular levels of NE and 5-HT. Chronic venlafaxine administration (14 days, 10 mg/kg, s.c.) elicited significant increases in cortical NE and 5-HT while producing a non-significant trend to increase cortical levels of normetanephrine. Additional studies revealed that combining normetanephrine with venlafaxine (10 mg/kg, i.p.), at a dose of normetanephrine (10 mg/kg, i.p.) that did not produce changes in extracellular levels of NE on its own, potentiated antidepressant-induced increases in extracellular NE. We also report mouse behavioral data involving the tail suspension test that complement the neurochemical observations. These preclinical findings, taken together, suggest that inhibiting both uptake 1 and uptake 2 via venlafaxine and normetanephrine, respectively, elicits a greater increase in cortical levels of NE than inhibiting either transporter alone.