Pronuclear microinjection remains the most widely used method for the germline modification of mice and other species. The method is conceptually quite simple and at least in rodents can produce transgenic offspring with relatively high efficiency. Here, we describe the various components of the production of transgenic mice including a detailed list of materials and equipment. We outline in detail the preparation of animals, the retrieval, culture and transfer of embryos, the preparation of DNA, and the microinjection process. We have added a substantial collection of notes with helpful suggestions that reflect the many years of experience we have using this technology and our continuing efforts to improve animal welfare and the efficiency of producing transgenics.