A series of experiments examined behavioural and autonomic aspects of stress and anxiety in rats subjected to either: (1) electrolytic lesions of the infralimbic cortex subregion of the medial prefrontal cortex; (2) electrolytic lesions of the prelimbic cortex subregion of the medial prefrontal cortex; (3) sham lesions of infralimbic or prelimbic cortex (sham control); or (4) no lesions (control). In exploration-based models of anxiety, infralimbic- or prelimbic-lesioned rats spent less time in the centre of an open field and less time on the exposed arms of an elevated plus maze, indicating increased anxiety. Locomotor activity was normal in the lesioned rats when tested in a non-stressful enclosed environment. In a step-down passive avoidance task, infralimbic-lesioned rats stepped down more quickly than controls onto a grid floor where they had been shocked 24 h previously. Prelimbic-lesioned rats were no different to controls on this test, although they showed greater latencies to step down onto the grid floor during conditioning. In a final experiment, indirect calorimetry was used to show that both infralimbic- and prelimbic-lesioned rats have essentially normal alterations in oxygen consumption and energy substrate utilisation when exposed to brief footshock. Thus, the impaired passive avoidance in infralimbic-lesioned rats cannot be attributed to decreased nociception. It is concluded that both the prelimbic and infralimbic regions play a role in anxiety, and that this role may be subtly differentiated. In particular, the infralimbic cortex may have a specific role in mediating the inhibition of behaviours associated with aversive outcomes.