In the absence of germ-line-competent ES cells in the rat, pronuclear microinjection remains an essential tool to generate transgenic rat models. However, DNA microinjection procedures first developed for mouse do not provide scientists with satisfying results when applied to rat. Here we describe optimized procedures for rat with a special focus on rat embryo production, in vitro incubation and culture, DNA pronuclear injection, and the transfer of embryos into foster females.