Dopaminergic and GABAergic neurons were visualized in the same section of rat substantia nigra (SN) and zona incerta (ZI) by a two-color double immunoperoxidase procedure or by double immunofluorescence. Rabbit antiserum to tyrosine hydroxylase (TH) and sheep antiserum to glutamic acid decarboxylase (GAD), markers for catecholaminergic and GABAergic neurons, respectively, were used as primary antisera. These techniques rely on species difference of primary antisera and non-crossreactivity of linking antisera. In normal and colchicine pretreated rats, SN pars compacta (SNC), SN pars lateralis (SNL), and ZI pars medialis (area A13) contained high densities of TH-positive neurons. Relatively few TH-positive cells were scattered in SN pars reticulata (SNR) and ZI pars lateralis (ZIL). In normal rats, GAD-positive boutons were more numerous throughout SNR and ZIL than in SNC, SNL, and area A13. In colchicine pretreated rats, the majority of neurons in SNR and ZIL and few neurons in SNC, SNL, and area A13 were GAD-positive and TH-negative. This study suggests a dichotomy of both SN and ZI into a predominantly dopaminergic and a predominantly GABAergic part.