The present study is an immunohistochemical analysis utilizing a series of mono- and polyclonal antibodies to myoglobin, desmin and vimentin in smooth and striated control muscle tissues, 7 alveolar and 7 embryonal rhabdomyosarcomas and 1 adult and 1 fetal rhabdomyoma with ultrastructurally proven rhabdomyoblastic differentiation in all the tumors. Formaldehyde-fixed and paraffin-embedded tissue was used for the immunohistochemical analysis of all the tumors, while ethanol fixation was also used for the analysis of the control tissues. The staining for myoglobin with the poly- and monoclonal antibody used was positive in both formaldehyde- and ethanol-fixed skeletal and cardiac control muscle. Trypsin treatment abolished the positive staining when the monoclonal antibody was used. Both the striated and smooth control muscle tissues were positively stained by the antidesmin antibodies. The influence of the fixative that was used and the trypsin treatment depended on the antibody used and the type and origin of the muscle tissue. All the tumors were positively stained with the polyclonal antimyoglobin and 8/14 rhabdomyosarcomas and the 2 rhabdomyomas were positively stained with the monoclonal antimyoglobin. All the tumors were positively stained with the polyclonal and 3 of the 5 monoclonal antidesmin antibodies used. Well-differentiated tumor cells were usually positively stained for both myoglobin and desmin. There were small, poorly differentiated tumor cells in the rhabdomyosarcomas and the fetal rhabdomyoma which were positively stained for desmin, whereas very few or no such cells were positively stained for myoglobin. A varying number of mostly small, poorly differentiated tumor cells were positively stained for vimentin in 12 of 14 rhabdomyosarcomas and in the fetal rhabdomyoma. The study showed that one of the monoclonal antidesmin antibodies produced the most consistent result with a positive staining in all cases. The monoclonal antimyoglobin antibody, which is a specific marker of rhabdomyoblastic differentiation, is also considered to be of value, although it did not produce positivity in all cases. It remains to be shown whether desmin can help in the diagnosis of poorly or undifferentiated rhabdomyosarcomas without light- or electron-microscopic evidence of rhabdomyoblastic differentiation.