The Brn-3 family of POU (Pit-Oct-Unc) homeodomain transcription factors regulate differentiation of neuronal cell types. The transcriptional activator Brn-3a is expressed in Ewing's sarcomas, which also express characteristic chimaeric proteins as a consequence of fusion of the TET family gene EWS to one of several ETS genes. We have previously demonstrated a physical interaction between Brn-3a and EWS proteins, and show here that the C-terminal POU domain but not N-terminal activation domain of Brn-3a can interact in vitro with the RNA-binding domain of EWS. Likely due to POU domain homology, the related factor Brn-3b can also interact with EWS, but to a lesser extent than Brn-3a. Importantly, Brn-3a but not Brn-3b interacts in vitro with chimaeric EWS/Fli-1, EWS/ATF-1 and EWS/ERG proteins. Furthermore, overexpression of EWS/Fli-1 but not EWS or Fli-1 inhibits Brn-3a-associated growth arrest and neurite outgrowth in neuronal cells, and specifically inhibits Brn-3a-dependent activation of p21 and SNAP-25 transcription. In contrast, upregulation of Bcl-2 expression and inhibition of apoptosis by Brn-3a is antagonized more by EWS than by EWS/Fli-1. These data demonstrate that oncogenic rearrangement of EWS to produce EWS/Fli-1 may enhance the antiapoptotic effect of Brn-3a and inhibit its ability to promote neuronal differentiation.