Previously, we generated thymic lymphoma cell lines from EbetaR/Rp53-/- (EP) double mutant mice where the T cell receptor (TCR) beta enhancer (Ebeta) was deleted, and the p53 gene was inactivated. Here, we characterized the EP cell lines to study the roles of the Ebeta and p53 on TCRbeta rearrangements during lymphomagenesis. Recombination activation genes (RAGs) were expressed, while the TCRbeta chain was not expressed in the EP cell lines. Dbeta-Jbeta rearrangements were not detected at all, and Dbeta1 and Dbeta2 cleavages were also not detected in the EP cell lines. However, Jbeta cleavages suppressed in Ebeta mutant thymocytes were readily detected in the EP cell lines. The Jbeta cleavages appeared to be uncoupled, aberrant, RAG-dependent and Ebeta-independent and were not detected in a p53 or Ebeta single mutant background, suggesting that the Jbeta cleavages are selected in the Ebeta and p53 double mutant background. Sequence analysis showed that the cleavage occurred in the cryptic recombination signal sequences (RSSs) present throughout Jbeta gene segments. The results implicate that the uncoupled and aberrant V(D)J cleavages may contribute to double-strand break-mediated genome instability during lymphomagenesis in EP mice.