Interferon-α therapy is associated with a high rate of depression, but the pathophysiological mechanisms remain unclear. The purpose of the present study was to investigate the effects of i.p. administered interferon-α on monoaminergic neurotransmission in the brain. The levels of monoamines and associated metabolites were measured in various regions of the rat brain using a high-performance liquid chromatography–electrochemical detection system. The serotonin transporter mRNA levels were also measured using in situ hybridization. After 1 day, dopamine turnover was diminished in the cortex. Norepinephrine turnover was decreased in most regions tested after 4 days. However, these changes were transient. After 14 days, serotonin turnover was increased in the frontal cortex and hippocampus in rats given a dose of 20 000 IU/kg; in the frontal cortex, hippocampus, amygdala, thalamus, hypothalamus and brainstem in those on 200 000 IU/kg; and in the thalamus and hypothalamus in those on 2 000 000 IU/kg (all P < 0.05). However, 14-day treatment did not significantly change serotonin transporter mRNA levels. Next, the question of whether interferon-α affects monoamine levels via induction of nitric oxide (NO), was investigated. However, there were no changes in either NO2– or NO3–, as markers of NO production, in any brain regions after 14-day treatment. These results suggest that chronic peripheral administration of interferon-α induces metabolic changes in the central serotonin system. Further investigation is needed to determine exactly how this cytokine affects the central serotonin system and to assess whether a central serotonin abnormality is involved in interferon-induced depression.