Immunocytochemical techniques were employed to investigate the normal adult organization, development, and effects of both neonatal and adult eye removal upon the organization of the serotoninergic projection to the hamster's superior colliculus. Immunocytochemistry, both alone and in combination with retrograde tracing with true blue and fluorogold, was used to determine the organization of the serotoninergic projection to the superior colliculus in normal adult hamsters. Immunoreactive fibers were present in all laminae of the superior colliculus, but they were most dense in the lower part of the stratum griseum superficiale, the stratum opticum, the stratum griseum profundum, and the stratum album profundum. When retrograde tracing from the colliculus was combined with immunocytochemistry for serotonin, cells containing both labels were found in the lateral portions of the nucleus raphe dorsalis and also in periaqueductal gray, the median and pontine raphe nuclei, and in the region of the medial lemniscus. Such cells were visible both ipsilateral and contralateral to the injection site. Serotonin immunoreactive fibers were visible in the superior colliculus by embryonic day 14 (2 days prior to birth). On the day of birth, a small number of immunoreactive fibers were present just below the pial surface and others were generally oriented either parallel or orthogonal to the collicular laminae. At this age, there were also many immunoreactive fibers that crossed from one side of the brainstem to the other in the commissure of the superior colliculus. Some serotonin-positive axons appeared to terminate as growth cones in fetal and newborn hamsters. Over the next 2 weeks, the serotoninergic innervation of the SC increased in density and assumed the laminar distribution observed in adult animals. By this age, only a very few immunoreactive fibers were present in the commissure of the superior colliculus. Removal of one eye on either on the day of birth or in adulthood resulted in reorganization of the serotoninergic innervation of the partially deafferented colliculus. There was a marked increase in the density of serotonin-positive fibers in the upper stratum griseum superficiale on the side ipsilateral to the remaining eye. This change was apparent within 2 weeks after enucleation in either neonatal or adult animals. Quantitative analysis demonstrated that the magnitude of this effect was greater after adult enucleation than after neonatal eye removal. Both neonatal and adult enucleation also resulted in an increase in the density of the serotoninergic projection to the dorsal and ventral lateral geniculate nuclei ipsilateral to the remaining eye.