The WT1 gene encodes a transcription factor implicated in normal and neoplastic development. The purpose of this study was to evaluate the diagnostic utility of a commercial WT1 antibody on a variety of pediatric small round blue cell tumors (SRBCT). A mouse monoclonal antibody (clone: 6F-H2, DAKO) raised against the N-terminal amino acids 1-181 of the human WT1 protein was tested. Microscopic sections from 66 specimens were stained using an antigen retrieval protocol with trypsin. The tumors included peripheral neuroectodermal tumors (PNET/Ewing's), neuroblastomas, desmoplastic small round cell tumors (DSRCT), lymphomas, Wilms' tumors, and rhabdomyosarcomas (RMS). One RMS case was investigated by Western blot analysis and RT-PCR to confirm the antibody specificity. A strong cytoplasmic staining was demonstrated in all RMS (11/11). The Western blot analysis confirmed the WT1 protein in the tissue, and the RT-PCR confirmed the presence of WT1 mRNA in the peripheral blood and tissue of one RMS patient. The Wilms' tumors had a variable nuclear and/or cytoplasmic positivity in most (17/24) cases. All PNET/Ewing's were negative. The nuclei of two lymphoblastic lymphomas stained strongly. A weak nuclear or cytoplasmic staining was reported in a few DSRCT (3/5), lymphomas (2/10), and neuroblastomas (2/8). This is a useful antibody in the differentiation of RMS from other SRBCTs. A strong cytoplasmic staining favors an RMS, and a strong nuclear staining is suggestive of a Wilms' tumor. A role for WT1 in the pathogenesis of rhabdomyosarcomas is raised. The limited sampling precludes any conclusions regarding the value of tissue or peripheral blood analysis for WT1 mRNA in patients with rhabdomyosarcoma.