The presynaptic mechanisms involved in the initiation of cocaine reinforcement were investigated using neurotoxin lesions. Rats were trained to intracranially self-administer cocaine (50 to 90 pmol) into the medial prefrontal cortex and after dose-effect analyses were completed, each rat received a unilateral 6-hydroxydopamine lesion (4 micrograms in 0.2 microliter) at the self-administration site. The lesion selectively decreased dopamine content in the medial prefrontal cortex (-45%) and decreased cocaine-maintained responding to vehicle levels. Lever-pressing could be reinstated by substituting dopamine (300 pmol) but not serotonin for cocaine. Dopamine self-administration was attenuated by including equimolar concentrations of the D2 dopaminergic antagonist sulpiride in the injectate. These results suggest that the initiation of reinforcing neuronal activity in the medial prefrontal cortex appears to result in part through the direct interaction of cocaine with presynaptic reuptake sites associated with dopaminergic nerve endings. The resulting increased synaptic concentration of the neurotransmitter may then interact with postsynaptic D2 binding sites to activate neuronal systems involved in the mediation of this reinforcement.