Objective: Dopaminergic neurons of the midbrain play an important role in reward-based learning and have been associated with a number of neurological diseases including Parkinson's disease and schizophrenia. An important regulator of the activity of midbrain dopaminergic neurons is the small conductance calcium activated potassium channel SK3 which plays a permissive role in the generation of burst activity associated with increased dopamine release. The aim of this study was to gain further insight into SK3 channel function and regulation in dopaminergic neurons by localization of the channel at a subcellular level in adult rat substantia nigra. Methods: The subcellular localization of SK3 channels in substantia nigra dopaminergic neurons was addressed by immunohistochemistry and confocal microscopy on adult rat brain sections in combination with biochemical rat brain membrane fractionation and Western blotting. Results: Immunohistochemistry and Western blotting experiments demonstrated a strong expression of SK3 channels in adult rat substantia nigra. Further localization studies revealed that SK3 channels are primarily expressed by dopaminergic neurons of the substantia nigra whereas little or no expression is detected in association with parvalbumin positive neurons. In dopaminergic neurons, the channel is primarily expressed in the somatodendritic area, where it displays a clustered localization. Markers of inhibitory and excitatory synapses revealed little overlap with SK3 channel expression suggesting a primarily extrasynaptic localization of the channel. Biochemical fractionation experiments confirmed this observation with rat midbrain SK3 signals being strongest in the P2 fraction though the protein was also present at lower intensity in the S1, LP1, PSD and P3 fractions. Conclusion: We find that SK3 channels are primarily extrasynaptically expressed on the soma and dendrites of adult rat substantia nigra dopaminergic neurons.