The neurotransmitter phenotype(s) of nigral neurons innervating the superior colliculus (SC) in the rat was examined using a combination of immunohistochemical techniques and fluorescent retrograde tracing. After double-immunofluorescent histochemistry for tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD), single cells in the rostral ventrolateral portion of the substantia nigra pars reticulata (SNr) and to a lesser extent the substantia nigra pars lateralis (SN1) displayed immunoreactivity to both antigens. Furthermore, following True blue (TB) injections into the SC and incubation for both TH and GAD immunoreactivity, a considerable number of cells in the SNr retrogradely labeled with TB (approximately 10%) were also immunopositive for both synthetic enzymes. The present study provides evidence for the coexistence of TH and GAD and thus, the coexistence of dopamine and GABA in a subpopulation of single nigrotectal projection cells.