Currently, the participation of neuropeptides in the generation of aversive states in the dorsal periaqueductal gray matter (DPAG) is poorly understood. The elevated plus maze (EPM) is widely used for studying the neurobiological mechanisms of anxiety in the laboratory. One difficulty with this test has been to evaluate the involvement of GABA mechanisms in the DPAG substrates of aversion, because microinjections of GABA receptor blockers in this region cause an intense behavioral activation. In this study, we examined in the EPM the effects of semicarbazide, a drug that acts indirectly on GABA neurotransmission through inhibition of the glutamic acid decarboxylase, and substance P (SP) following microinjections into the dorsal periaqueductal gray. Semicarbazide caused a clear decrease in the number of entries and time spent in the open arms. These results confirm previous data showing that GABA has a modulatory role in the DPAG, probably through reduction of tonic inhibitory mechanisms on neural substrates of aversion. A similar pattern of behavioral responses was observed with SP. However, these effects were more pronounced with intermediate doses of SP (25 ng), confirming the characteristic bell-shaped dose-effect function of this neuropeptide. The proaversive effects observed with DPAG microinjections of SP in the present study gain further relevance when combined with previous reports that have shown unconditioned and conditioned aversive effects following DPAG microinjections of SP in other animal models of aversion.