Immunohistochemistry remains the current ancillary method of choice in the pathologic evaluation of small blue round-cell tumors. In at least 20% of cases of rhabdomyosarcoma (RMS), it is considered an essential factor in the final and/or differential diagnosis of the malignancy. Newer immunostains (antimyogenin, MyoD1) generated against intranuclear myogenic transcription factors offer pathologists the best hope for improving the sensitivity and specificity of RMS diagnosis. A large series of RMS (956) were studied consecutively from the intergroup rhabdomyosarcoma study and children's oncology group files, along with multiple other malignant, benign or reactive lesions. A panel of antibodies to muscle-related antigens (myogenin, MyoD1, desmin, muscle-specific actin) was studied using formalin-fixed, paraffin-embedded tissue, an avidin-biotin/peroxidase complex immunohistochemical technique, antigen retrieval technique as appropriate, and automated immunostaining. Myogenin and MyoD1 were equally sensitive (positive for 97% of RMS cases), with both also showing similar specificity (90% vs. 91% of cases) for the diagnosis of RMS. Myogenin and MyoD1 staining were sometimes intact in areas of coagulative tumor necrosis, but negated by B5 fixation. Isolated, rare benign myogenin-positive nuclei were seen infrequently in reactive lymph nodes. Specifically, both myogenin and MyoD1 had significantly greater extent of expression for alveolar RMS (ARMS) than embryonal RMS (ERMS) (both with P < 0.001). Similarly, both myogenin (P = 0.001) and MyoD1 (P < 0.001) had significantly higher expression for ARMS than RMS, not otherwise specified (NOS). They were never expressed in undifferentiated sarcomas; however, reactive or regenerative myocytes did show expression. Immunostains against intranuclear myogenic transcription factors are, at present, the best available markers for confirming the diagnosis of RMS. Their differential expression in reactive myogenic lesions, variability in ARMS versus ERMS, and absence in undifferentiated sarcomas suggest new biologic questions to be explored in future studies.