Although dopamine has been considered as the only neurotransmitter in the nigrostriatal pathway, studies carried out in the last two decades have suggested the existence of a nondopaminergic nigrostriatal projection, and more recently, gamma-aminobutyric acid (GABA) has been identified as its neurotransmitter. In this study, we used the combination of immunocytochemistry for tyrosine hydroxylase (TH; a marker of dopaminergic neurons), in situ hybridization (ISH) for two different isoforms of glutamic acid decarboxylase (GAD65 and GAD67, the rate-limiting enzyme in GABA synthesis) and retrograde tracing techniques to investigate the possible existence of nigrostriatal neurons containing both neurotransmitters (dopamine and GABA) in the rat. Our results revealed that approximately 10% of mesostriatal dopaminergic neurons, most of them lying in the medial region of the substantia nigra pars compacta (SNC) and neighbouring A10 region, contain GAD65 mRNA. These findings reveal a third nigrostriatal pathway formed by dopaminergic/GABAergic neurons. Contrasting with the idea that in the basal ganglia, dopamine and GABA are released from different cell populations, the results suggest a more complex dopamine/GABA interaction than previously assumed, probably including cotransmission.