Human skeletal muscle differentiation and maturation follows a precise sequence of events. To investigate whether and to what extent rhabdomyosarcoma (RMS) cells follow a comparable sequence, 29 fresh frozen specimens of RMS (14 primary and 15 relapses) were immunostained with antibodies directed against developmentally regulated myosin heavy chains (MHC), ie, fetal, fast, and slow MHC, in addition to desmin and vimentin. Four distinct patterns of expression were observed: I) RMS cells expressing exclusively vimentin and desmin (n = 7), II) in addition to expression of vimentin and desmin, a minority of neoplastic cells were immunoreactive with fetal MHC (n = 6), III) in addition to pattern II, fast MHC was expressed (n = 7), and IV) RMS cells simultaneously expressing vimentin, desmin, fetal, fast, and slow MHC (n = 9). Accordingly, the proportion of the MHC immunoreactive RMS cells increased gradually along with the four patterns of expression evolving from less than 25% up to 75% for fetal MHC, from less than 25% up to 50% for fast MHC, and up to 25% for slow MHC in the last category. Vimentin and desmin were coexpressed by almost all RMS cells. Double immunostaining revealed that comparable with the myogenic cells in the developing fetal skeletal muscle, expression of fetal MHC could be demonstrated in the same neoplastic cells either in conjunction with fast or slow MHC. In contrast, only in RMS, slow MHC expression in conjunction with fast MHC could be observed in the neoplastic cells. Neither the shape or size of neoplastic RMS cells, nor the histopathological types, nor tumor localization were related to the expression pattern of developmentally regulated MHC (fetal, fast, and slow MHC). These results confirm the commitment of the RMS cells to the myogenic pathway and demonstrate a restricted and aberrant differentiation pattern of the neoplastic cells in RMS compared with normal myogenesis, independent of histopathological types of RMS.