Distinction of rhabdomyosarcomas (RMS) from other tumors can be difficult, requiring use of monoclonal antibodies against unique muscle proteins. In previous studies using cryostat sections, we found that MyoD1, a transcription factor that activates the expression of muscle-specific genes, is highly expressed in rhabdomyosarcoma and useful in diagnosis. Recent studies using paraffin sections demonstrated poorer results, leading us to compare MyoD1 staining using dual paraffin versus frozen sections of tumor tissue. We performed immunostaining on companion frozen and paraffin sections of 69 pediatric tumors (40 rhabdomyosarcomas and 29 putative nonrhabdomyosarcomas [nRMS]), using similar dilutions of anti-MyoD1 monoclonal antibody 5.8A. Twenty-four of 40 (60%) frozen rhabdomyosarcomas and 14 of 40 (35%) paraffin-embedded rhabdomyosarcomas were nuclear positive for MyoD1. Of the 29 putative nonrhabdomyosarcomas, six of 29 (21%) and three of 29 (10%) were nuclear positive, using frozen and paraffin sections, respectively. These positive cases included one ectomesenchymoma, one embryonal sarcoma of liver, one neurofibroma, three undifferentiated sarcomas, and one Wilms' tumor. In retrospect, these nine nuclear-positive cases appear to have myogenic potential. In six frozen and 12 paraffin RMS sections and in nine frozen and 15 paraffin putative nRMS sections, only nonspecific cytoplasmic staining was observed. We conclude that MyoD1 immunostaining of small round cell tumors is more reliable and sensitive using frozen sections, despite its potential applicability in paraffin sections.