We describe a rapid method for initial localization of protein/DNA-binding sites in large DNA segments, even when biological information is limited. The procedure combines the gel retardation assay with bidirectional deletion analysis using restriction enzymes. Electrophoresis of binding reactions with a bidirectional set of progressively shortened probes results in a stairway pattern of both uncomplexed DNA and specific protein/DNA complexes. The loss of binding upon deletion of specific DNA segments localizes the boundaries of protein/DNA interaction sites. Stairway assays are illustrated using cloned DNA fragments spanning three histone gene promoters, but it is possible to adapt this method for any segment of genomic DNA that can be amplified using PCR methods. Multiple binding sites were established for transcription factors, chromatin-associated proteins and sequence specific nuclear matrix proteins, thereby validating this approach for at least three classes of DNA-binding proteins.