Electrophysiological recordings from identified synapses in CNS slice preparations in vitro provide important information regarding the connectivity of neuronal circuits and the underlying cellular mechanisms responsible for neuronal excitability and synaptic transmission. We present an anatomical, electrophysiological, and pharmacological characterization of a novel brain slice preparation (BLA-mPFC) to investigate basolateral amygdala synaptic input to rat layer V medial prefrontal cortex pyramidal neurons. A fluorescent tracer (DiI) unilaterally infused in vivo into the basolateral amygdala was used to detect amygdala efferent fibers innervating layer V of the prelimbic and infralimbic cortices within prefrontal cortex slices. In vitro, evoked synaptic responses elicited by stimulating identified basolateral amygdala pathway terminals within the acute BLA-mPFC slice preparation yielded monosynaptic excitatory postsynaptic responses in layer V pyramidal neurons from the prelimbic cortex as determined by extracellular and intracellular recordings. The BLA-mPFC preparation provides essential knowledge of amygdaloid input to the medial prefrontal cortex where information from various brain areas is integrated and returned to subcortical structures, such as the amygdala itself. In addition to investigating normal synaptic function, this preparation provides opportunities to investigate this synapse in animals which have received drugs chronically or have been manipulated genetically to model specific mental diseases known to involve prefrontal cortex and/or amygdala pathology (e.g., schizophrenia, addiction, anxiety, and depression).