Prompt and accurate diagnosis of small round cell tumors warrants ancillary studies. Recently, two-color fluorescence in-situ hybridization (FISH) using probes for specific gene rearrangements has gained wide acceptance. EWS gene rearrangements, present in essentially 100% of Ewing's Sarcoma/peripheral primitive neuroectodermal tumor, were evaluated by FISH on frozen sections (FS) of tumor biopsies from 10 patients, plus a negative control, and in seven other malignant neoplasms of childhood. 4mu FS were hybridized overnight, using a single EWS gene-specific probe spanning the EWS breakpoint. We identified EWS rearrangements in 8 of 10 cases (80%) of Ewing's Sarcoma/pPNET. There are no known false positives in diploid or near-diploid tumors, or in any of the non-EWS tumors tested; the uncommon false negative can be confirmed by RT-PCR. Hyperdiploid cases with multiple copies of chromosome 22 may be better evaluated by two-color FISH. This is the first use on FS biopsy material of a single probe for EWS, capable of detecting all known EWS rearrangements, in ES and other tumors. Utilization of this ancillary technique on FS for ES/pPNET and other tumors with distinctive chromosomal translocation is highly specific, reliable, expeditious (24-36 hours) and cost-effective.