It is widely known that hypocretins are essential for the regulation of wakefulness. Our recent reports have found that hypocretin-1 shows a direct postsynaptic excitatory effect on rat prefrontal cortex (PFC) pyramidal neurons. It remains unclear whether hypocretin-1 may interact with two classical neurotransmitter systems, glutamate and gamma-aminobutyric acid (GABA) in rat PFC. For this reason, we here investigated the modulatory actions of hypocretin-1 with these two transmitters on freshly isolated PFC pyramidal neurons using whole-cell patch-clamp recordings. We found that coadministration of hypocretin-1 and glutamate showed a synergistic effect on the recorded cells, and hypocretin-1 could excite the neurons even if GABA was present. Thus, our data suggest that there may be hypocretin-glutamate and hypocretin-GABA interactions in the PFC.