Initial effects of drugs of abuse seem to converge on the mesolimbic dopamine pathway originating from the ventral tegmental area (VTA). Many drugs of abuse, already after a single dose, are able to modulate the glutamatergic transmission activating the VTA dopamine neurons, which could represent a critical early step in the development of addiction. Ligands acting on the benzodiazepine site of the inhibitory gamma-aminobutyric acid type A (GABA-A) receptors are known to be rewarding in animal models and have abuse liability in humans, but notably little evidence exists that the mesolimbic dopamine system would be involved in their effects. We have now found that, similarly to classical drugs of abuse like morphine and ethanol, single in vivo doses of benzodiazepine-site agonists induce a modulation in the glutamatergic transmission of VTA dopamine neurons. This was seen 24 h after the injection as an increase in the ratio between AMPA and NMDA receptor-mediated excitatory currents using whole-cell patch-clamp configuration in mouse VTA slices. The effect was due to increased frequency of spontaneous miniature AMPA receptor-mediated currents. It lasted at least 3 days after the injection of diazepam, and it was prevented by co-administration of the benzodiazepine-site antagonist flumazenil or the NMDA receptor antagonist dizocilpine. Also a single injection of the GABA-A receptor alpha-1 subunit-preferring benzodiazepine-site ligand zolpidem produced an increase in the AMPA/NMDA ratio in VTA dopamine neurons. These findings suggest a role for the mesolimbic dopamine system in the initial actions of and on neuronal adaptation to benzodiazepines.