The distribution of serotonin immunoreactivity in the superior colliculus (SC) of the rat, hamster, chipmunk, cat, and monkey was studied using a sensitive immunohistochemical method. In all of these animals, serotonin immunoreactivity formed a dense network of varicose fibers throughout the SC. These fibers had a characteristic arrangement corresponding to the laminar structures of the SC. Except in the chipmunk, serotonergic fibers were more dense in the stratum griseum superficiale than in the other layers. In the SC of the chipmunk, these fibers appeared evenly distributed. To explore the degree of scrotonergic innervation in each layer, a semi-quantitative assay of serotonin immunoreactive varicosities was conducted in the rat, chipmunk, cat, and monkey. Peaks in varicose density were seen in the stratum griseum superficiale, the stratum griseum intermedium and the stratum griseum profundum. In the rat, cat, and monkey, the highest density of these varicosities was in the stratum griseum superficiale. On the other hand, the stratum griseum intermedium of the chipmunk SC received the greatest innervation of serotonergic varicose fibers.