Background: Reversal learning is widely used to analyze cognitive flexibility and characterize behavioralabnormalities associated with impulsivity and disinhibition. Recent studies using fMRI have focused onregions involved in reversal learning with negative and positive reinforcers. Although the frontal cortexhas been consistently implicated in reversal learning, few studies have focused on whether reward andpunishment may have different effects on lateral frontal structures in these tasks. Here, in eight healthysubjects, we used functional near infra-red spectroscopy (fNIRS) to characterize brain activity dynamicsand differentiate the involvement of frontal structures in learning driven by reward and punishment.Results: We observed functional hemispheric asymmetries between punishment and reward processingby fNIRS following reversal of a learned rule. Moreover, the left dorsolateral prefrontal cortex (l-DLPFC)and inferior frontal gyrus (IFG) were activated under the reward condition only, whereas the orbito-frontalcortex (OFC) was significantly activated under the punishment condition, with a tendency towardsactivation for the right cortical hemisphere (r-DLPFC and r-IFG). Our results are compatible with thesuggestion that the DLPFC is involved in the detection of contingency change. We propose a newrepresentation for reward and punishment, with left lateralization for the reward process.Conclusions: These results provide insights into the indirect neural mechanisms of reversal learning andbehavioral flexibility and confirm the use of fNIRS imaging in reversal-learning tasks as a translationalstrategy, particularly in subjects who cannot undergo fMRI recordings.