Histologic examination was carried out in 65 cases of childhood rhabdomyosarcoma (RMS), 53 embryonal, and 12 alveolar. Cross-striations were seen on light microscopy in 12 (23%) embryonal and 4 (33%) alveolar tumors. The capacity of immunohistochemical staining (PAP technique) to increase diagnostic accuracy was assessed, using antibodies against myoglobin, the MM isoenzyme of creatine kinase, desmin, calcium magnesium-dependent ATPase of sarcoplasmic reticulum and calsequestrin. Myoglobin was detected in 16 (30%) embryonal and eight (67%) alveolar RMS, higher numbers than obtained by viewing cross-striations on light microscopy. The creatine kinase antibody was slightly better than the antibody to myoglobin and 15 of 25 (60%) embryonal RMS were positive when both specificities were used. The remaining three antibodies were less useful. Of 13 (two alveolar and 11 embryonal) RMS studied by electron microscopy, four showed cross-striations, contained late myoblasts, and were positive for myoglobin. Three additional cases showed only late myoblasts and one of these was positive for myoglobin. Thus, 16 of 25 (64%) of the embryonal and seven of nine (78%) of the alveolar RMS showed either positive immunostaining or ultrastructural features of RMS. This study indicates that a combination of immunohistochemical staining, using antimyoglobin and anticreatine kinase (MM isoenzyme) antibodies, and electron microscopy are useful markers in the diagnosis of childhood RMS.