The processes of making transgenic animals by microinjecting DNA into the pronucleus of a fertilized oocyte or after the transfection of embryonic stem cells are now well established. However, attempts have also been made, with varying degrees of success, to use spermatozoa as a vector for transgenesis in mammals and other vertebrates during the last decade. A number of different approaches for making transgenic spermatozoa have been developed. These include directly incubating mature, isolated spermatozoa with DNA or pretreating mature, isolated spermatozoa before assisted fertilization. Microinjection procedures have also been established to transfect male germ cells directly in vivo within the seminiferous tubules or to reimplant previously isolated male germ cells submitted to in vitro transfection into a recipient testis. The latter two techniques present the advantage of being able to create transgenic progeny simply by mating with wild-type females, which avoids the possibility of interference or damage as a result of assisted fertilization or the manipulation of embryos. The different aspects of sperm-mediated transgenesis are presented.