Most Ewing tumors (ET), including Ewing sarcomas, peripheral primitive neuroectodermal tumors (PNET), and Askin's tumors, can be defined according to the specific chromosomal translocations t(11;22)(q24;q12) (EWS-FLI-1) or t(21;22)(q21;q12) (EWS-ERG). Detection of the chimeric RNA transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) has greatly facilitated the diagnosis of ET. Because of variable chromosomal breakpoint locations, however, the EWS gene fusions with FLI-1 and ERG genes are highly heterogenous, resulting in different sizes of the amplification products. To improve the diagnostic usefulness of the RT-PCR assay, we have developed an assay to detect chimeric mRNA transcripts by nested RT-PCR, followed by digestion of the PCR fragments with three different restriction endonucleases. This allows confirmation of the specificity of the PCR product and provides a rapid method to determine the combination of exons present in a transcript. In the 12 Ewing tumors tested, five different exon combinations were detected. In nine repeat biopsies of four patients, the case-specific translocation remained unchanged. One additional central PNET had no ET-specific translocation. In conclusion, the suggested combination of RT-PCR and restriction analysis of the PCR products allows a rapid and specific determination of ET-specific translocations.