In vivo microdialysis in freely moving rats was used to monitor novelty-evoked elevations in extracellular dopamine in the nucleus accumbens septi (NAS) and to examine the role of the ventral subiculum of the hippocampus and glutamatergic transmission in the ventral tegmental area (VTA) on these elevations. Exposure to novel stimuli evoked investigatory activity and increased nucleus accumbens dopamine. Unilateral injections of the sodium channel blocker tetrodotoxin (0.16 ng/0.5 µL) into the ventral subiculum ipsilateral to the dialysed NAS abolished novelty-evoked elevations in dopamine. Injections of tetrodotoxin into the contralateral VS did not prevent novelty-evoked elevations in nucleus accumbens dopamine. Unilateral perfusion (via microdialysis) of the ionotropic glutamate receptor antagonists kynurenic acid (1 mm) into the ipsilateral but not the contralateral VTA blocked novelty-evoked elevations in nucleus accumbens dopamine. Neither unilateral injections of tetrodotoxin nor unilateral perfusion of kynurenic acid disrupted investigatory behaviour. These data indicate that phasic elevations in nucleus accumbens dopamine evoked by exposure to unconditioned novel stimuli are dependent on impulse flow from the hippocampus and glutamatergic transmission in the VTA.