The distribution and morphology of adenosine deaminase, substance P, leucine-enkephalin, corticotropin-releasing factor, and calcitonin gene-related peptidelike immunoreactive cells and fibers throughout the superior colliculus of the rat were examined by means of the unlabelled-antibody peroxidase-antiperoxidase method. Adenosine deaminase immunoreactive cells were found in the stratum opticum and lower stratum griseum superficiale; substance P immunoreactive cells were localized to the upper stratum griseum superficiale, and calcitonin gene-related peptide immunolabelled neurons were situated in deeper strata. Substance P, leucine-enkephalin, and calcitonin gene-related peptide immunoreactive fibers were distributed similarly in their lamination and in their patchlike organization. Corticotropin-releasing factor immunoreactive fibers were observed evenly throughout all the strata and were fewer in the stratum griseum superficiale. These findings suggest that, as in afferent modules and segregated efferents of the mammalian superior colliculus, the cells and fibers containing neuroactive substances and neuroactive substance-related enzymes also show a segregated and laminar distribution.