The chromosomal translocation specifically linked to the Ewing sarcoma family results in the generation of fusion proteins comprising the amino terminal portion of EWS and the DNA-binding domain of ets transcription factors. The EWS/ets chimeric proteins act as aberrant transcription factors leading to tumorigenic processes. We searched for genes specifically activated in Ewing sarcoma cells but not in other tumor cell lines using the gene array technique, and found significantly enhanced expression of the Id2 gene. High levels of Id2 transcripts were detected in Ewing sarcoma cell lines and tumor tissues. The EWS/ets chimeric proteins activated the Id2 gene via the 5'-upstream promoter sequence. Chromatin-immunoprecipitation revealed a direct interaction of EWS/Fli-1 with the promoter regions of the Id2, TGF-beta type II receptor, cyclin D1, and c-myc genes. Since EWS/Fli-1 transactivates c-myc, a cooperative action of the chimeric protein and c-myc leads to overexpression of Id2. In the present study, we suggest that Id2 is a target of the chimeric proteins and that the c-myc/Id2 pathway plays a pivotal role in the tumorigenic processes provoked by EWS/ets proteins.