Objective: The pathophysiology of schizophrenia has been linked to increased density and occupancy of dopamine D2 receptors (D2R) in the striatum. To explore the pathophysiological consequences of enhanced D2R function, transgenic mice were generated that reversibly overexpress D2Rs selectively in striatal medium spiny neurons (Kellendonk et al., 2006, Neuron 49:603-15). These mice exhibited impairments in prefrontal cortical function, including working memory, accompanied by an increase in dopamine tissue content in medial prefrontal cortex (mPFC). Methods: To investigate the cellular basis of this prefrontal dopamine dysregulation, we combined retrograde tracing experiments with electrophysiological and immunohistochemical techniques in D2R overexpressing (OE) and control mice. In vitro patch clamp analyses of retrogradely labeled DA ventral tegmental area (VTA) neurons were performed 3 weeks after selective stereotactic injection of fluorescent tracer beads into the mPFC of adult (4-8 month old) mice. After recording, neurobiotin-filled cells were immunostained for tyrosine hydroxylase to verify their neurochemical identity. Results: Synaptically isolated mesocortical DA VTA neurons from D2R OE mice showed significantly faster pacemaker frequencies compared to control littermates (D2R OE: f=5.0±0.6 Hz (n=13); control: f=3.2±0.4 Hz (n=11); p=0.026). This enhancement of spontaneous electrical activity was not present in nigrostriatal DA neurons (D2R OE: f=2.3±0.3 Hz (n=4); control: f=2.6±0.1 Hz (n=4); p=0.550). Similar to the working memory deficit, the elevated mesocortical excitability persisted after doxycycline-induced suppression of D2R transgene expression in adulthood (D2R OE: f=5.4±0.6 Hz (n=6); control: f=3.7±0.5 Hz (n=10); p=0.042). Conclusion: Increased excitability of mesocortical DA neurons may be a novel candidate mechanism involved in the dopamine dysregulation in prefrontal cortex and the associated deficits in prefrontal function in schizophrenia.