Derangements of the prefrontal cortex (PFC) and of brainstem monoaminergic systems occur in depression and schizophrenia. Anatomical and functional evidence supports a PFC control of the brainstem monoaminergic systems. Similarly, the PFC contains a high density of monoamine receptors for which antipsychotic drugs exhibit high affinity. This raises the possibility that pathological or drug-induced changes in PFC may subsequently alter monoaminergic activity. Recent data indicate that a substantial proportion of PFC pyramidal neurons projecting to the ventral tegmental area (VTA) or the dorsal raphe nucleus (DR) express the 5-HT2A receptor mRNA, which suggests that atypical antipsychotic drugs affect serotonergic and dopaminergic function by targeting PFC 5-HT2A receptors. Using electrophysiological and tract-tracing techniques we examined whether PFC pyramidal neurons projecting to DR are segregated from those projecting to the VTA. Sequential electrical stimulation of these nuclei in anaesthetized rats evoked antidromic potentials from both areas in the same pyramidal neurons of the medial PFC (60%, n=30). A similar percentage of dual DR+VTA projection neurons (50%) was obtained using the reciprocal collision test (n=85). Similarly, tracer application (Fluoro-Gold in VTA and cholera toxin B in DR, or vice versa) retrogradely labelled pyramidal neurons in PFC projecting to VTA (81±18), to DR (52±9) and to both nuclei (31±4, n=5 rats). Overall, these results indicate that the PFC may simultaneously coordinate the activity of dopaminergic and serotonergic systems within a short temporal domain, supporting a concerted modulation of the ascending serotonergic and dopaminergic activity during antipsychotic drug treatment.