A novel method was developed to prepare for PCR- mediated DNA footprinting walking map. This method is based on the amplification of the cleaved strands using multi-specific primers to walk along the whole DNA template. At first, long enough target DNA fragment without being labeled is incubated in the presence or absence of nuclear proteins and cleaved randomly on average once by either a chemical reagent or an enzyme such as DNaseⅠ. Then the labeled multi-specific primers (sense or antisense) are utilized to amplify the cleaved DNA template to produce labeled single strands which can walk along the whole template. At last, the single strand DNA is separated by electrophoresis in a denaturing polyacrylamide gel, followed by autoradiography analysis. The result is the appearance of a “gap” in the sequence ladder of protein-combined DNA compared to the pattern of unbound or unprotected DNA. This method can footprint walking along any length of DNA template in one time theoretically if enough specific primers are utilized. This method was applied to investigate the partial footprinting map of 5′ flanking region from －1190 to －273 of human stem cell factor gene.