Electroporation has been the method of election for transfection of murine embryonic stem cells for over 15 years; however, it is a time consuming protocol because it requires large amounts of DNA and cells, as well as expensive and delicate equipment. Lipofection is a transfection method that requires lower amounts of cells and DNA than electroporation, and has proven to be effi cient in a large number of cell lines. It has been shown that after lipofection, mouse embryonic stem cells remain pluripotent, capable of forming germ line chimeras and can be transfected with greater effi ciency than with electroporation; however, gene targeting of mouse embryonic stem cells by lipofection has not been reported. The objective of this work was to fi nd out if lipofection can be used as effi ciently as electroporation for regular gene targeting protocols. This context compares gene targeting effi ciency between these techniques in mouse embryonic stem cells E14TG2a, using a gene replacement type vector. No differences were found in gene targeting effi ciency between groups; however, lipofection was three times more effi cient than electroporation in transfection effi ciency, which makes lipofection a less expensive alternative method to produce gene targeting in mouse embryonic stem cells.