Rho GTPase controls multiple signal-transduction pathways involving the actin cytoskeleton and the microtubule cytoskeleton in processes such as the cell cycle, morphogenesis, and cell migration. The activity of Rho GTPases, such as Rac1, Cdc42, and RhoA, is regulated by GTPase-activating proteins (GAPs), guanine nucleotide exchange factors (GEFs), and guanine nucleotide dissociation inhibitor (GDI). The GEFs activate Rho GTPases through exchange of GDP for GTP. A group of the GEF family, CDM-GEF, containing the Dock180 Homology Region 2 (DHR2) domain is subdivided into four groups based on amino acid sequences. One of these subgroups, DOCK-A, includes DOCK1, DOCK2, and DOCK5, and activates Rac1 Rho GTPase. Mouse Dock5 (mDock5) is structurally similar to DOCK1 and composed of 1868 amino acids containing Src homology 3 (SH3), DHR1, DHR2, and a proline-rich (PR) motif. We generated an mDock5-specific antibody, which detected denatured and nascent forms of mDock5. We determined tissue-specific expression patterns and subcellular localization of mDock5 using this antibody. This antibody provides a way to delineate the biological functions of mDock5 in vivo.