The present status of genomic DNA subtraction techniques is reviewed. The advantages and disadvantages of the widely-used methods of genome subtraction are discussed. Using the kinetic model of subtractive hybridization developed by us previously (Sverdlov and Ermolaeva, 1993; Sverdlov and Ermolaeva, 1994), the application of genome subtraction to various problems is analyzed. It is concluded that the technique should be further advanced based on subtraction of single-stranded DNAs. This strategy would enable one to efficiently extract target sequences omitting the stage of genome simplification.