We have employed immunohistochemical and computerized morphometric procedures to study substance P-containing neurons in the tegmentum of adult humans. An estimated 192, 500±40,500 substance P-containing neurons were found in three main cytoarchitectural regions: the mesencephalic reticular formation, the central gray, and the pontine reticular formation. The morphology of the immunoreactive neurons varied according to the region in which they were found. On the basis of size alone two types of substance P-containing neurons, large and small, were readily distinguishable by eye and measurement. Within each of the three main regions it was possible to distinguish distinct subgroups using cell size, morphology and position. Large neurons were concentrated in the caudal midbrain (pedunculopontine tegmental nuclei), in the oral pontine reticular nucleus and in the lateral dorsal tegmental nucleus. In contrast, small neurons were concentrated in the rostral mesencephalic reticular formation (cuniform nuclei). Both small and large neurons were found in the midbrain and pontine raphe nuclei. In addition, small neurons were concentrated in discrete midline regions (the periaqueductal gray, the tegmental nuclei of the pontine central gray, and the interpeduncular nucleus). The findings suggest that the majority of neurons in the brainstem tegmental nuclei previously identified as cholinergic also contain substance P in humans.