EWS and related TAFII68 and TLS/FUS genes are fused with different genes encoding transcription factors in various human cancers. The products of these genes have the ability to bind RNA and have been shown to be part of splicing and transcription complexes. We show that the EWS, TAFII68 and TLS/FUS proteins are expressed to various levels in all adult murine tissues. We characterize a new isoform of EWS that is specifically expressed in the central nervous system, in both mice and humans. It is shown to be related to a splice variant which includes a new 18-bp exon, termed 4', between exon 4 and 5. The detection of this isoform in spontaneously differentiating SH-SY5Y neuroblastoma cells and in nerve growth factor-induced PC12 cells further links this isoform to neural differentiation. RT-PCR experiments indicate that the level of expression of the brain-specific EWS isoform is stable during brain development whereas that of the ubiquitous EWS isoform decreases during this period. The two isoforms show a parallel decrease in expression after birth. The 4' exon is not detected in tumour-specific EWS fusion transcripts, suggesting that its presence may impair their oncogenic properties. Interestingly, sequences of the 4' exon and flanking regions show remarkable similarities to that of the neural-specific c-src exon, suggesting common mechanisms for the alternative splicing of these exons. The phylogenetic conservation and relationship to neural differentiation strongly suggests an important functional role for this exon.