The Tol2 transposable element is a powerful genetic tool in model vertebrates and has been used for transgenesis, insertional mutagenesis, gene trapping, and enhancer trapping. However, an in vivo transposition system using Tol2 has not yet been developed. Here we report the in vivo Tol2 transposition system in a model vertebrate, zebrafish. First, we constructed transgenic zebrafish that carried single-copy integrations of Tol2 on the genome and injected transposase mRNA into one-cell stage embryos. The Tol2 insertions were mobilized efficiently in the germ lineage. We then mobilized an insertion of the Tol2 gene trap construct in the nup214 gene, which caused a recessive lethal mutant phenotype, and demonstrated that this method is applicable to the isolation of revertants from a transposon insertional mutant. Second, we constructed transgenic fish carrying the transposase cDNA under the control of the hsp70 promoter. Double-transgenic fish containing the transposase gene and a single-copy Tol2 insertion were treated with heat shock at the adult stage. We found that transposition can be induced efficiently in the male germ cells. We analyzed new integration sites and found that the majority (83%) of them were mapped on chromosomes other than the transposon donor chromosomes and that 9% of local hopping events mapped less than 300 kb away from the donor loci. Our present study demonstrates that the in vivo Tol2 transposition system is useful for creating genome-wide insertions from a single-copy donor and should facilitate functional genomics and transposon biology in vertebrates.