This protocol introduces the technique of homologous recombination in bacteria to insert a linear DNA fragment into bacterial artificial chromosomes (BACs). Homologous recombination allows the modification of large DNA molecules, in contrast with conventional restriction endonuclease-based strategies, which cleave large DNAs into numerous fragments and are unlikely to permit the precise targeting afforded by recombination-based approaches. The method uses a phage lambda-derived recombination system (using exo, beta, and gam) as well as other enzymatic activities provided by the host (Escherichia coli). In the method described here, a DNA fragment encoding enhanced cyan fluorescent protein is inserted immediately after the start codon of the gene encoding choline acetyltransferase (“ChAT”), the final enzyme in acetylcholine biosynthesis, using homologous recombination between sequences that are present both on the introduced DNA fragment and in the target BAC. The desired recombination products are identified via positive selection for resistance to kanamycin. In principle, the resulting modified BAC could be used to produce transgenic mice that express this fluorescent protein in cholinergic neurons. The approach described here could be used to insert any DNA fragment.