This study introduces a novel and highly efficient galK-based positive/negative selection system for bacterial artificial chromosome (BAC) recombineering. The system allows for the modification of BACs without introducing unwanted selectable markers at the modification site. Three new recombineering strains (SW102, SW105, and SW106) were developed, each containing a complete galactose operon except for a precise deletion of the galK gene. These strains also carry inducible Cre or Flp genes, enabling the introduction of point mutations, deletions, and loxP sites into BAC DNA. The galK selection system is more efficient than other selection methods and reduces background due to spontaneous mutations. The system uses galK for both positive and negative selection, allowing for the rapid introduction of desired modifications into BAC DNA. The galK selection cassette is small (around 1200 bp) and easy to amplify by PCR and introduce into bacteria using electroporation. The system was tested for its ability to introduce point mutations, deletions, and loxP sites into BAC DNA, and it was shown to be effective in generating conditional targeting vectors for mouse ES cell experiments. The system also allows for the removal of unwanted genes from BACs through BAC trimming, which is useful for positional cloning experiments. The study demonstrates the utility of the galK selection system for BAC modification and highlights its efficiency and simplicity compared to other methods. The system is also applicable for generating conditional targeting vectors and for studying the effects of gene deletions on gene regulation. The strains developed in this study should be useful for genetic manipulation not only of BACs but also of the E. coli genome itself.This study introduces a novel and highly efficient galK-based positive/negative selection system for bacterial artificial chromosome (BAC) recombineering. The system allows for the modification of BACs without introducing unwanted selectable markers at the modification site. Three new recombineering strains (SW102, SW105, and SW106) were developed, each containing a complete galactose operon except for a precise deletion of the galK gene. These strains also carry inducible Cre or Flp genes, enabling the introduction of point mutations, deletions, and loxP sites into BAC DNA. The galK selection system is more efficient than other selection methods and reduces background due to spontaneous mutations. The system uses galK for both positive and negative selection, allowing for the rapid introduction of desired modifications into BAC DNA. The galK selection cassette is small (around 1200 bp) and easy to amplify by PCR and introduce into bacteria using electroporation. The system was tested for its ability to introduce point mutations, deletions, and loxP sites into BAC DNA, and it was shown to be effective in generating conditional targeting vectors for mouse ES cell experiments. The system also allows for the removal of unwanted genes from BACs through BAC trimming, which is useful for positional cloning experiments. The study demonstrates the utility of the galK selection system for BAC modification and highlights its efficiency and simplicity compared to other methods. The system is also applicable for generating conditional targeting vectors and for studying the effects of gene deletions on gene regulation. The strains developed in this study should be useful for genetic manipulation not only of BACs but also of the E. coli genome itself.