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Publisher Summary This chapter focuses on highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. Fish are excellent candidates for the production of transgenics for two important reasons. First, fish represent the largest and most diverse group of vertebrates and provide an advantageous system for in vivo studies of developmental processes to gain knowledge of gene regulation and the action of gene products in vertebrates. Second, conventional selective breeding of fish for improved growth or other characteristics is a very slow process. Transgenic fish technology has the potential to improve genetic traits such as increased growth potential, disease resistance, improved feed conversion efficiency, or other desirable genetic traits for aquaculture in one generation. The establishment of methods for successful transgenesis is one of the basic criteria for an organism to be referred to as model organism. Several endonucleases (meganucleases) encoded by introns and inteins have been shown to promote homing (lateral transfer) of their respective genetic elements into intron- or inteinless homologous allelic sites. By introducing sitespecific double-strand breaks (DSBs) in intronless alleles, these nucleases create recombinogenic ends that engage in gene conversion, resulting in duplication of the intron. The I-SceI system has been used as a tool in mammalian cells and Drosophila. The meganuclease can also be used in fish for comparative studies of cis-acting regulatory elements and homologous recombination (HR). Meganuclease transgenesis as described in this chapter could be further improved, including insulators upstream and downstream of the DNA of interest to protect the transgene from the influences of heterochromatin and epigenetic control of surrounding genomic sequences.