HOX11 is a homeobox-containing oncogene of specific T-cell leukemias. We determined the DNA binding specificity of the Hox11 protein by using a novel technique of random oligonucleotide selection developed in this study. The optimal Hox11 binding sequence, GGCGGTAAGTGG, contained a core TAAGTG motif that is consistent with a prediction based on the residues at specific positions that potentially make DNA base contacts and models of homeodomain-DNA interaction proposed from studies with other homeodomains. The specific interaction between Hox11 and the selected optimal binding sequence was further confirmed by band-shift and DNA competition assays. Given that the Hox11 homeodomain shares low homology with other well studied homeodomains, the presence of a predictable recognition core motif in its optimal binding sequence supports the notion that different homeodomains interact with DNA in a similar manner, through highly conserved residues at specific positions that allow contact with DNA.