In the intact brain, dopamine midbrain neurons signal novelty or unexpected reward and punishment by a change of their firing pattern: they switch from a low frequency pacemaker mode to a transient high frequency burst mode. This burst mode is essential to generate phasic release of dopamine in axonal target areas and in turn behavioral activation via low-affinity D1 receptors. By combining in vivo single unit recordings and juxtacellular labeling in wildtype and K_ATP channel (Kir6.2) knockout mice, we show that functional K-ATP channels are necessary for in vivo burst firing selectively in dopamine neurons in the medial substantia nigra. In contrast, in vivo burst firing is unaffected by the lack of K_ATP channels in dopamine neurons in the lateral substantia nigra and the ventral tegmental area. Virally-mediated cell-selective silencing by expression of negative dominant Kir6.2 subunits demonstrated that postsynaptic K_ATP channels in dopamine neurons are essential for in vivo burst firing. Moreover, selective silencing of K_ATP channels in medial substantia nigra neurons was sufficient to reduced novelty-induced behaviors like enhanced locomotion or rearing. These data suggest a surprising in vivo function for K_ATP channels in a subpopulation of dopamine neurons where they facilitate high-frequency burst discharge and behavioral activation. This in vivo function also sheds new light on the established pathophysiological role of K_ATP channels in the selective vulnerability of dopamine neurons in the substantia nigra (Liss et al. 2005, Nat. Neuroscience), which might be due to their novel role of enhancing in vivo excitability and thus their potential to contribute to excitotoxicity.