Genomic deletions induced by imprecise excision of transposons have been used to disrupt gene functions in Drosophila. To determine the excision properties of Tol2, a popular transposon in zebrafish, we took advantage of two transgenic zebrafish lines Et(gata2a:EGFP)pku684 and Et(gata2a:EGFP)pku760, and mobilized the transposon by injecting transposase mRNA into homozygous transgenic embryos. Footprint analysis showed that the Tol2 transposons were excised in either a precise or an imprecise manner. Furthermore, we identified 1093-bp and 1253-bp genomic deletions in Et(gata2a:EGFP)pku684 founder embryos flanking the 5' end of the original Tol2 insertion site, and a 1340-bp deletion in the Et(gata2a:EGFP)pku760 founder embryos flanking the 3' end of the insertion site. The mosaic Et(gata2a:EGFP)pku684 embryos were raised to adulthood and screened for germline transmission of Tol2 excision in their F(1) progeny. On average, ∼42% of the F(1) embryos displayed loss or altered EGFP patterns, demonstrating that this transposon could be efficiently excised from the zebrafish genome in the germline. Furthermore, from 59 founders, we identified one that transmitted the 1093-bp genomic deletion to its offspring. These results suggest that imprecise Tol2 transposon excision can be used as an alternative strategy to achieve gene targeting in zebrafish.