The Ewing's sarcoma family of tumors (ESFT) contains a characteristic translocation the chimeric transcript of which is translated to become the EWS−FLI1 fusion protein. EWS−FLI1 regulates transcription and posttranscriptional splicing. Elimination of EWS−FLI1 protein from ESFT cells induces apoptosis and reduces xenograft tumor growth. Therefore the production of a biologically active recombinant EWS−FLI1 could lead to discoveries that would enhance our mechanistic understanding of ESFT. We have cloned, expressed, and purified a biologically active recombinant EWS−FLI1 in Escherichia coli using affinity column chromatography. A refolding procedure was required to render the recombinant EWS−FLI1 soluble in relatively native conditions. The structural alterations induced by the refolding procedure were monitored by SDS-gel electrophoresis, circular dichroism, and steady-state fluorescence spectroscopy. Recombinant EWS−FLI1 under native conditions approaches a largely unfolded conformation. Recombinant EWS−FLI1 protein under native conditions specifically binds to DNA and transcribes RNA. Our biologically active recombinant EWS−FLI1 oncoprotein will be useful to identify functional molecular partners and inhibitors.