The differentiation of extraocular muscles was studied immunohistochemically in externally normal human embryos (Carnegie stages 13-23), using antibodies to carbonic anhydrase (CA) III and β-enolase as the markers of type 1 and type 2 muscle fibers, respectively. At stage 18, some myoblasts were immunoreactive to β-enolase antibodies, however, CA-III immunoreactivity was not observed around the optic vesicle. At stage 20, CA-III immunoreactivity appeared in some muscle fibers of extraocular muscles. From stage 21 to stage 23, CA-III-immunoreactive fibers increased and almost equalled the number of β-enolase-immunoreactive fibers. These findings suggest that CA-III-immunoreactive type 1 fibers appear in the late stage of myogenesis compared with β-enolase-immunoreactive type 2 fib