Conditional mutagenesis in mice is a key approach in neurosciences that makes it possible to investigate the functions of defined genes within certain neural subpopulations. The approach is based on the combination of transgenic expression of a recombinase with targeted candidate genes containing recognition sequences of this recombinase generated by gene targeting. Here we describe three major recombination systems, the Cre/loxP, Flp/FRT, and Dre/rox, and present a procedure that allows the generation of target alleles for these systems. We provide detailed protocols for the culture and transfection of embryonic stem (ES) cells and for the identification and confirmation of homologous recombinants. This chapter also provides an overview of recent developments such as gene targeting in oocytes and discusses future perspectives.