A DNA binding protein was identified which binds to two novel target-like sequences: (i) at the 5' flanking site of the breakpoint junction of chromosome 8 in a patient with T-acute lymphoblastic leukemia (ALL) carrying the t(8;14)(q24;q11) rearrangement and (ii) on chromosome 1 in three of five T-ALL patients with the t(1;14)(p32;q11) rearrangement. This protein [provisionally called recombination hotspot associated factor (ReHF-1)] was also found to bind to a similar target sequence that is present immediately at the 3' end of the human V delta 3 gene segment. In a small number of lines tested, the ReHF-1 protein was expressed in gamma delta lineage T cells and in a number of B cell precursor ALLs whose TCR delta locus has been rearranged. The molecular weight of ReHF-1 protein was determined to be approximately 30 kDa by UV cross-linking analysis. Gel filtration chromatography and sedimentation velocity centrifugation analyses indicate that the ReHF-1 protein exists as a multimeric protein in its native form. These data might suggest a possible role for this protein in the rearrangement of the TCR delta locus. Furthermore, another protein, ReHF-2, that appears to have strict sequence specificity was found to bind only to the complementary single strand of the target sequence. The interaction of these proteins with a conserved target sequence at the chromosomal breakpoint junction might suggest that they are involved in a novel enzymatic mechanism reminiscent of the general features of DNA recombination or replication events in Escherichia coli or Saccharomyces cerevisiae.