Alternative splicing of the dopamine D2 receptor gene produces two distinct isoforms referred to as D2long (D2L) and D2short (D2S). In mesencephalic dopamine neurons, inhibition of the firing rate through activation of somatodendritic D2 receptors and blockade of neurotransmitter release through stimulation of terminal D2 receptors represent major roles of D2 autoreceptors. Recently, data obtained from D2L-deficient mice suggested that D2S acts as the preferential D2 autoreceptor. In the present study, we investigate whether this D2 isoform-specific autoreceptor function is linked to differences in the subcellular localization and/or signaling properties of the D2S and D2L using mesencephalic neurons transfected with enhanced green fluorescent protein (EGFP)-tagged receptors. Our results show that EGFP-tagged D2S and D2L are localized to the axonal and somatodendritic compartments of mesencephalic neurons. In addition, we demonstrate that EGFP-tagged D2S and D2L regulate cellular excitability, neurotransmitter release and basal levels of intracellular calcium with similar effectiveness. Overall, our morphological and electrophysiological studies suggest that the major D2 autoreceptor function attributed to D2S is likely explained by the predominant expression of this isoform in dopamine neurons rather than by distinct subcellular localization and signaling properties of D2S and D2L.