(1) A Little information exists on the distribution of Slit-Robo-GTPase activating proteins (srGAPs), particularly about their intracellular locations, which may provide further clues to their functions. The purpose of this study is to elucidate the expression patterns of the three srGAPs in wild-type rat brains at adult and various developmental stages, and in the cultured cortical neurons. (2) Immunohistochemical method was applied to detect the distribution and localization of the srGAPs in the normal rat brains at adult and various developmental stages, and in the cultured cortical neurons using the rabbit polyclonal antibodies. (3) Immunohistochemical analysis demonstrated that the three srGAPs were mainly expressed in neurons throughout the brain. More importantly, srGAPs translocated during development by a highly regulated shuttling process between the nucleus and the cytoplasm of neurons and their expression patterns were not overlapping. In cultured cortical neurons srGAPs were found in equal amounts in the cytoplasm, nucleus, in neurites, and growth cones. When neurons were maintained in vitro for longer time, the amount of srGAPs in the nucleus strongly increased. (4) These results suggest that srGAPs are not only involved in the regulation of the Slit-Robo signal transduction, but also in neuronal development and that the translocation of srGAPs is important for their functions.