A new procedure for the construction of linker scanning mutants is described. A plasmid containing the target DNA is randomly linearized and slightly shortened by a novel combination of established methods. After partial apurination with formic acid a specific nick or small gap is introduced at the apurinic site by exonuclease III, followed by nuclease S1 cleavage of the strand opposite the nick/gap. Synthetic linkers are ligated to the ends and plasmids having the linker inserted in the target DNA are enriched. Putative linker scanning mutants are identified by their topoisomer patterns after relaxation with topoisomerase I. This technique allows the distinction of plasmids differing in length by a single basepair. We have used this rapid and efficient strategy to generate a set of 32 linker scanning mutants covering the chicken lysozyme promoter from -208 to +15.