Cognitive flexibility, the ability to change the pattern of behaviour in the face of changing environmental conditions, is crucial for survival and is thus associated with many psychiatric disorders. To asses and study cognitive flexibility in different species many tasks have been developed including reversal learning paradigms. Reversal learning paradigms use a set of different stimuli which are in turn rewarded or not rewarded. They have pointed out important brain regions for the control of cognitive flexibility. Prefrontal cortex and its subregions – orbitofrontal cortex and medial prefrontal cortex with connections to the striatum and amygdala are implicated in cognitive flexibility. Therefore, we wanted to explore the role of prefrontal cortex subregions and site-specific serotonin signalling in cognitive flexibility using touchscreen-based serial reversal behavioural paradigm in rat models. We performed local pharmacological manipulations using intracranial microinfusions of GABAergic and glutamatergic agents as well as serotonin receptor antagonists. The behavioural testing provided us with some important insights. We showed that there is a functional dissociation between medial and lateral orbitofrontal cortex, which not only seems to have separate, but also opposite functions and is important for the inhibition of responses and previously learned behaviour. The medial prefrontal cortex is also implicated in cognitive flexibility. However, specific serotonin signalling modulation failed to produce any significant results and thus needs some further investigation.