N-[[1-(5-fluoropentyl)-1H-indazol-3-yl]carbonyl]-3-methyl-D-valine methyl ester (5F-ADB) is one of the most potent synthetic cannabinoids and elicits severe psychotic symptoms in humans, sometimes causing death. To investigate the neuronal mechanisms underlying its toxicity, we examined the effects of 5F-ADB on midbrain dopaminergic and serotonergic systems, which modulate various basic brain functions such as those in reward-related behavior. 5F-ADB-induced changes in spontaneous firing activity of dopaminergic and serotonergic neurons were recorded by ex vivo electrophysiological techniques. In dopaminergic neurons, 5F-ADB (1 μM) significantly increased the spontaneous firing rate, while 5F-ADB failed to activate dopaminergic neurons in the presence of the CB1 antagonist AM251 (1 μM). However, the same concentration of 5F-ADB did not affect serotonergic-neuron activity. These results suggest that 5F-ADB activates local CB1 receptors and potentiates midbrain dopaminergic systems with no direct effects on midbrain serotonergic systems.