The Ewing family of tumors is recurrently characterized at the molecular level by the presence of a fusion transcript between the EWS gene on chromosome 22 and either the FLI-I or ERG genes, 2 closely related members of the Ets family of transcription factors. We have investigated 12 primary human tumors, 11 xenografts and 11 cell lines, which have been shown to express chimeric EWS transcripts in search of p53 mutations. Fragments of exons 5 to 8 and the corresponding consensus splice sequences were amplified by PCR and analyzed by denaturing gradient gel electrophoresis (DGGE). In 12 of 34 samples p53 mutations were detected (including 4 samples with multiple p53 mutations). The distribution of the mutations in the various samples was as follows: primary tumors 2/12; cell lines 5/11; xenografts 5/11. No correlation between the presence or absence of p53 mutations and the presence of a specific EWS chimeric transcript was observed. In addition, we observed that p53 mutations were almost always associated with a second hit (either deletion or second mutation) on the other p53 allele.