Anatomical and physiological studies in the rat have shown projections from the medial dorsal thalamus to the anterior cingulate cortex. We used multibarrel iontophoresis to identify the neurotransmitter used in this thalamic projection. Extracellular responses were recorded from 165 cingulate neurons in anesthetized rats after electrical stimulation of the medial dorsal thalamus and vicinity. Forty-four of these cells (27%) showed an excitatory response to thalamic stimulation. In a further 40 cells that showed no baseline excitation, iontophoresis of the GABAA antagonist bicuculline methiodide revealed excitatory responses. The GABAB antagonist CGP-35348 attenuated longer-latency inhibition in 5 of 10 cells. In 23 of 49 (47%) of the above cells, AMPA antagonist iontophoresis (either CNQX or DNQX) selectively decreased the excitatory response to thalamic stimulation. The NMDA antagonist 3[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid had no such effect. These data suggest that the thalamic projection to anterior cingulate cortex is glutamatergic, acting principally via AMPA receptors, and that the response of cingulate neurons to thalamic stimulation is regulated by GABA acting at both GABAA and GABAB receptors.