DNA binding specificity of the RBP-J_x protein was extensively examined. The mouse RBP-J_x protein was originally isolated as a nuclear protein binding to the J_x type V(D)J recombination signal sequence which consisted of the conserved heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences separated by a 23-base pair spacer. Electrophoretic mobility shift assay using DNA probes with mutations In various parts of the J_x recombination signal sequence showed that the RBP-J_x protein recognized the sequence outside the recombination signal in addition to the heptamer but did not recognize the nonamer sequence and the spacer length at all. Database search identified the best naturally occurring binding motif (CACTGTGGGAA-CGG) for the RBP-J_x protein in the promoter region of the m8 gene in the Enhancer of split gene cluster of Drosophila. The binding assay with a series of m8 motif mutants indicated that the protein recognized mostly the GTGGGAA sequence and also interacted weakly with ACT and CG sequences flanking this hepta-nucleotide. Oligonucleotides binding to the RBP-J_x protein were enriched from a pool of synthetic oligonucleotides containing 20-base random sequences by the repeated electrophoretic mobility shift assay. The enriched ollgomer shared a common sequence of CGTGGGAA. All these data indicate that the RBP-J_x protein recognizes a unique core sequence of CGTGGGAA and does not bind to the V(D)J recombination signal without the flanking sequence.