Naturally occurring chromosomal fusion of the Ewings Sarcoma Oncogene (EWS) to distinct cellular transcription factors, produces aberrant transcriptional activators that function as dominant oncogenes. In Malignant Melanoma of Soft Parts the N-terminal region of EWS is fused to C-terminal region of the cAMP-inducible transcription factor ATF1. The EWS/ATF1 fusion protein binds to ATF sites present in cAMP-responsive promoters via the ATF1 bZIP domain and activates transcription constitutively in a manner that is dependent on an activation domain (EAD) present in EWS. To further define the requirements for trans-activation we have performed mutational analysis of EWS/ATF1 in mammalian cells and report several new findings. First, trans-activation by EWS/ATF1 does not require dimerisation with other ATF family members present in mammalian cells. Second, in contrast to the earlier suggestion of an allosteric role, the EAD can act directly. Third, determinants of trans-activation are dispersed throughout the EAD and cooperate synergistically to produce potent trans-activation. We also report that the region of EWS containing the EAD can activate transcription in Yeast. This latter finding might enable a genetic approach to understanding the mechanism of transcriptional activation by EWS and development of high-throughput screens for EWS inhibitors.