In Sprague-Dawley rats by means of in vivo microdialysis, we have shown that presentation to rats a conditioned chamber in which a ton was previously paired with footshock (acquisition of a conditioned fear response to the tone) produces an increase in extracellular levels of citrulline (an NO co-product) in the medial prefrontal cortex and results in freezing response (fear index). This increase was not observed in control rats (same procedure, no footshock) and it was prevented by infusions into the medial prefrontal cortex of N®-propyl-L-arginine (1 mM), a neuronal NO synthase inhibitor. The infusion of 1mM N®-propyl-L-arginine into the medial prefrontal cortex reduced freezing to the conditioned chamber, but did not affect movement activity of the rats. Data obtained indicate that the nitrergic system of the medial prefrontal cortex implicates in regulation of fear expression produced by contextual signals of danger.