INTRODUCTIONBreeding of axolotl (Ambystoma mexicanum) can be accomplished by natural or artificial methods. Although natural matings are a very easy way of obtaining axolotl embryos, in vitro fertilization can have other advantages. For example, through in vitro fertilization, it is possible to know the exact time of fertilization. This protocol describes preparation of a sperm suspension and in vitro fertilization of axolotl eggs after injection of females with follicle stimulating hormone (FSH).