ARBP is a nuclear protein that specifically binds to matrix/scaffold attachment regions (MARs/SARs). Here we characterize by DNase I footprinting, dimethyl sulfate protection, and mobility shift assays two binding sites for ARBP within a chicken lysozyme MAR fragment. Our results indicate that ARBP recognizes a novel DNA sequence motif containing the central sequence 5'-GGTGT-3' and flanking AT-rich sequences. Binding occurs through major groove contacts to two guanines of the central sequence. Collective and single-base substitutions in the 5'-GGTGT-3' core motif result in loss or significant reductions of ARBP binding, underscoring the importance of the GC-rich core sequence. Structural elements of the sequence motif are probably also recognized. The affinity of ARBP to both binding sites is surprisingly high [KD = (2-6) x 10(-10) M]. High-affinity recognition of the identified DNA motif in MARs/SARs by ARBP is likely an important feature in the domain organization of chromatin.