The results of this study support the conclusion that dopaminergic cells can be distinguished from non-dopaminergic cells, at both the light- and electron-microscopic level, by cytological features, and particularly by the pattern of Nissl substance. In both the substantia nigra and the ventral tegmental area, two main categories of cell type can be identified in Nissl preparations: (1) dark-staining, basophilic cells with large masses of Nissl substance and (2) light-staining cells with more translucent cytoplasm. The following findings provide evidence that the basophilic cells of both substantia nigra and ventral tegmental area are the dopaminergic cells. (1) There is a good correlation between the topographic distribution of basophilic cells and that of dopaminergic cells mapped by both histofluorescence and immunohistochemical methods. (2) After unilateral destruction of the dopaminergic neurons by intracerebral injection of 6-hydroxydopamine in the dopaminergic pathway, the basophilic cells in the substantia nigra and ventral tegmental area disappeared on the lesion side, while the lighter-staining cells appeared unaffected. (3) In normal rats, and in rats with unilateral 6-hydroxydopamine lesions, intraventricular injection of [3H]norepinephrine was used for specific labeling of dopaminergic neurons. In autoradiograms of semithin sections, such labeling was observed only in dark-staining and not in light-staining cells, and in cases of unilateral 6-hydroxydopamine lesion was totally absent on the lesion side. Electron-microscopy showed much of the cytoplasm of the basophilic dopaminergic cells to be densely filled with free ribosomes associated with large, well organized complexes of rough endoplasmic reticulum. The cytoplasm of the light, non-dopaminergic cells contains only sparse free ribosomes and small, widely spaced aggregates of rough endoplasmic reticulum. Both cell types occur in a similar variety of size and shape.