Transposon systems are widely used for generating mutations in various model organisms. PiggyBac (PB) has recently been shown to transpose efficiently in the mouse germ line and other mammalian cell lines. To facilitate PB's application in mammalian genetics, we characterized the properties of the PB transposon in mouse embryonic stem (ES) cells. We first measured the transposition efficiencies of PB transposon in mouse embryonic stem cells. We next constructed a PB/SB hybrid transposon to compare PB and Sleeping Beauty (SB) transposon systems and demonstrated that PB transposition was inhibited by DNA methylation. The excision and reintegration rates of a single PB from two independent genomic loci were measured and its ability to mutate genes with gene trap cassettes was tested. We examined PB's integration site distribution in the mouse genome and found that PB transposition exhibited local hopping. The comprehensive information from this study should facilitate further exploration of the potential of PB and SB DNA transposons in mammalian genetics.