Animals confronting threatening stimuli respond with a coordinated set of autonomic, neuroendocrine, neurochemical, and behavioral responses that constitute the stress response. The role of the NMDA receptor and its glycine modulatory site was investigated in a rat conditioned stress model. Behavioral, neuroendocrine, and neurochemical analyses were conducted. Regional dopamine (DA) and serotonin (5-HT) utilization was assessed by postmortem tissue measurements of metabolite-to-parent neurotransmitter ratios. Rats were conditioned to fear a tone previously paired with footshock. The following day, rats were systemically administered saline or the NMDA glycine site antagonist (+)-HA-966 before exposure to thirty minutes of conditioned stress. Conditioned stress resulted in a selective increase in medial prefrontal cortical DA and 5-HT utilization, elevation in serum corticosterone, and freezing behavior in control animals. The conditioned stress-induced increase in DA utilization in control animals was also detected in the lateral prefrontal cortex and nucleus accumbens, whereas DA utilization was not affected in the perirhinal or cingulate cortices, lateral-basolateral amygdaloid complex, anterior ventromedial caudatoputamen, or posterior dorsolateral caudatoputamen. Pretreatment with (+)-HA-966 at 15 mg/kg completely abolished the conditioned stress-induced increase in DA utilization in the medial and lateral prefrontal cortices. This effect was regionally specific since (+)-HA-966 pretreatment did not block increased DA utilization in the nucleus accumbens. This effect was also neurochemically specific since the stress-induced increase in 5-HT utilization in the medial prefrontal cortex was not affected by (+)-HA-966 pretreatment. Pretreatment with (+)-HA-966 did not affect stress-induced serum corticosterone elevation but did attenuate the freezing response. Control experiments demonstrated that (+)-HA-966 pretreatment did not (1) induce sedation, (2) interfere with habituation to a novel environment, (3) alter basal DA, 5-HT, or serum corticosterone levels, or (4) block acquisition of aversive memories. These data suggest that the NMDA receptor complex and associated glycine modulatory site may play an important role in the afferent control of the mesoprefrontal cortical DA system during conditioned stress. The relevance of these findings to schizophrenia and human anxiety disorders such as post- traumatic stress disorder are discussed.