DT diaphorase [NAD(P)H:quinone oxidoreductase] activity was measured in subcellular fractions from homogenates of striatum, frontal cortex, hippocampus, cerebellum, hypothalamus and substantia nigra. This flavoprotein, which by definition oxidizes dihydronicotinamide adenine dinucleotide and dihydronicotinamide adenine dinucleotide phosphate at equal rates and is completely inhibited by 10(-5) M dicoumarol, was found to constitute 80-90% of the total dihydronicotinamide adenine dinucleotide- and dihydronicotinamide adenine dinucleotide phosphate-reductase activities in all brain regions studied. Antibodies raised against purified cytosolic DT diaphorase from the rat liver cross-reacted with the brain enzyme and inhibited soluble DT diaphorase from striatum and cerebellum to 80-90%. Immunohistochemical studies with the same antibodies demonstrated the occurrence of DT diaphorase immunoreactivity in a population of neurons in the substantia nigra and ventral tegmental area. In some neurons there was a colocalization of DT diaphorase and tyrosine hydroxylase-like immunoreactivity. The dense network of DT diaphorase-immunoreactive fibres in the striatum disappeared along with the dopaminergic innervation after 6-hydroxydopamine lesion. DT diaphorase immunoreactivity was also found in Bergmann glia, astrocytes and tanycytes. No correlation appeared to exist between the localization of neuronal DT diaphorase immunoreactivity and the dihydronicotinamide adenine dinucleotide phosphate-diaphorase-like activity, as defined by tetrazolium salt staining, used as a marker for certain peptidergic and cholinergic neurons. However, in, for example, glial cells in the cerebellum, DT diaphorase might contribute or be responsible for the histochemical dihydronicotinamide adenine dinucleotide phosphate-diaphorase activity.