Dopaminergic axons innervating the prefrontal cortex (PFC) target both pyramidal cells and GABAergic interneurons. Many of these dopamine (DA) axons in the rat coexpress the peptide neurotransmitter neurotensin. Previous electrophysiological data have suggested that neurotensin activates GABAergic interneurons in the PFC. Activation of D2-like DA receptors increases extracellular GABA levels in the PFC, as opposed to the striatum, where D2 receptor activation inhibits GABAergic neurons. Because activation of presynaptic D2 release-modulating autoreceptors in the PFC suppresses DA release but increases release of the cotransmitter neurotensin, D2 agonists may enhance the activity of GABAergic interneurons via release of neurotensin. In order to determine if neurotensin can activate GABAergic interneurons, we treated rats with the peptide neurotensin agonist, PD149163, and examined Fos expression in PFC neurons. Systemic administration of PD149163 increased overall Fos expression in the PFC, but not in the dorsal striatum. PD149163 induced Fos in PFC interneurons, as defined by the presence of calcium-binding proteins, and in pyramidal cells. Pretreatment with the high-affinity neurotensin antagonist, SR48692, blocked neurotensin agonist-induced Fos expression. These data suggest that neurotensin activates interneurons in the PFC of the rat.