Recent experiments have provided valuable information about the functional organization of the subregions of the rat prefrontal cortex. This review assesses the findings from studies in which the role of the cholinergic and dopaminergic systems in the anterior cingulate, prelimbic, and infralimbic areas was investigated by using several different approaches—that is, chemical lesions, pharmacological manipulations, or in vivo microdialysis. Cholinergic and dopaminergic input to the prelimbic and infralimbic subregions may modulate multiple behavioral processes. Activation of the muscarinic cholinergic receptors or activation of the dopamine D1 receptors in the prelimbic and infralimbic areas modulates working memory processes. Cholinergic input to the prelimbic and infralimbic areas may also modulate attention, since changes in acetylcholine release occur when there is an increase in attentional demands. In addition, activation of dopaminergic receptors in the prelimbic and infralimbic areas influences behavioral flexibility. Cholinergic input to the anterior cingulate does not appear critical for working memory but likely contributes to attentional processes. Overall, the results suggest that acetylcholine and dopamine affect multiple behavioral functions subserved by the rat prefrontal cortex that depend on modulating activity in specific prefrontal cortex subregions.