S-100 protein has two subunits, S-100α and S-100β. S-100α is predominant in neurons and slow-twitch muscle fibers, while S-100β exists in glial cells and Schwann cells. In the present study, the presence of S-100α and S-100β was investigated immunohistochemically in the extraocular muscles of 15 human embryos (Carnegie stages 13 to 23) and in one fetus (10 weeks of gestation). During stages 13-17,no immunoreactivities to S-100α and S-100β were found around the optic vesicle. At stage 18,immunoreactivity to S-100β appeared around the optic vesicles and increased as the embryonic stages advanced. In the fetus, the nerve fibers in the extraocular muscles were slightly immunoreactive to S-100α, but the muscle fibers were not. These findings suggested that extraocular muscles are innervated in the late embryonic period, around stage 18.