This paper describes a novel and highly efficient method for modifying Bacterial Artificial Chromosomes (BACs) using galK selection. The authors developed three new recombinering strains (SW102, SW105, and SW106) that allow BACs to be modified using galK positive/negative selection. This two-step selection procedure eliminates the need for an unwanted selectable marker at the modification site. The galK gene, which encodes galactokinase, can be selected both for and against, reducing background and increasing efficiency compared to other selection methods. The small size of the galK cassette (around 1200 bp plus homology arms) makes it easy to amplify by PCR and introduce into bacteria using electroporation. The authors demonstrate how galK selection can be used to introduce point mutations, deletions, and loxP sites into BAC DNA, facilitating functional studies of SNPs and disease-causing mutations, identifying long-range regulatory elements, and constructing conditional targeting vectors. The method is efficient, requires minimal hands-on time, and can be applied to various BAC modifications, including BAC trimming and the construction of gene-targeting vectors.This paper describes a novel and highly efficient method for modifying Bacterial Artificial Chromosomes (BACs) using galK selection. The authors developed three new recombinering strains (SW102, SW105, and SW106) that allow BACs to be modified using galK positive/negative selection. This two-step selection procedure eliminates the need for an unwanted selectable marker at the modification site. The galK gene, which encodes galactokinase, can be selected both for and against, reducing background and increasing efficiency compared to other selection methods. The small size of the galK cassette (around 1200 bp plus homology arms) makes it easy to amplify by PCR and introduce into bacteria using electroporation. The authors demonstrate how galK selection can be used to introduce point mutations, deletions, and loxP sites into BAC DNA, facilitating functional studies of SNPs and disease-causing mutations, identifying long-range regulatory elements, and constructing conditional targeting vectors. The method is efficient, requires minimal hands-on time, and can be applied to various BAC modifications, including BAC trimming and the construction of gene-targeting vectors.