Rhabdomyosarcoma presents special diagnostic problems when it involves the uterine cervix in young children because tumor cells may lack marked atypia and may blend with the normal, immature, condensed, cellular stroma, rendering diagnosis difficult. Myogenic makers are a valuable ancillary technique for establishing a diagnosis of rhabdomyosarcoma. However, desmin positivity has been reported in cervical stromal cells, which can confound diagnosis. To determine whether immunohistochemical markers of skeletal muscle differentiation are helpful in the diagnosis of uterine botryoid rhabdomyosarcoma, we compared the immunohistochemical staining pattern of cervical rhabdomyosarcoma from 3 patients with that of normal uteri from age-matched autopsy controls by using antibodies for desmin, smooth muscle actin, muscle-specific actin, myoD1, myogenin, and WT-1. All tumors demonstrated at least focal immunopositivity for desmin, muscle-specific actin, smooth muscle actin, myoD1, and WT-1, and 1 tumor was also positive for myogenin. Autopsy controls showed only scattered subepithelial stromal immunoreactivity for desmin, muscle-specific actin, smooth muscle actin, and WT-1 and showed cytoplasmic, but not nuclear, immunopositivity for myoD1 and myogenin. Myometrium was diffusely positive for desmin and muscle-specific actin. We conclude that desmin, muscle-specific actin, smooth muscle actin, and WT1 are not specific for discriminating embryonal rhabdomyosarcoma from normal subepithelial cells in the female genital tract of children, although the number of immunopositive cells is consistently larger in rhabdomyosarcoma. Nuclear staining for myoD1 and myogenin appears not to occur in normal tissue, but it may be absent or sparse in embryonal rhabdomyosarcoma. Our findings indicate that, in this anatomic site, the diagnosis of rhabdomyosarcoma and in particular determination of tumor margins remain very reliant on histomorphology.