Monoamine reuptake inhibitors increase brain-derived neurotrophic factor (BDNF) activity, and this growth factor is regarded as an interesting target for developing new antidepressant drugs. The aims of this study were to evaluate whether monoaminergic reuptake inhibition increases BDNF in vivo and in vitro as predicted by the neurotrophic hypothesis of depression, and whether triple reuptake inhibition has a superior BDNF response compared to dual reuptake inhibition. Twenty-one days of oral treatment (30 mg/kg) with the dual serotonin/noradrenaline reuptake inhibitor duloxetine or the triple serotonin/noradrenaline/dopamine reuptake inhibitor DOV 216,303 restored BDNF protein levels in the rat hippocampus, which were initially decreased due to injection stress. The prefrontal cortex contained increased BDNF levels only after DOV 216,303 treatment. In vitro, neither duloxetine nor DOV 216,303 altered intracellular BDNF levels in murine HT22 neuronal cells. In contrast, BDNF release was more effectively decreased following treatment with DOV 216,303 in these cells. In rat C62B astrocytomas, both antidepressants increased intracellular BDNF levels at their highest nontoxic concentration. C62B astrocytomas did not release BDNF, even after antidepressant treatment. Increased BDNF levels support the neurotrophic hypothesis of depression, but our findings do not clearly evidence that the BDNF response after triple reuptake inhibitors is more effective than after dual reuptake inhibitors. Moreover, the data suggest that the role of BDNF in neurons and astrocytes is complex and likely depends on factors including specificity of cell types in different brain regions, cell–cell interactions, and different mechanisms of action of antidepressants used.