myr 5 is an unconventional myosin (class IX) from rat that contains a Rho-family GTPase-activating protein (GAP) domain. Herein we addressed the specificity of the myr 5 GAP activity, the molecular mechanism by which GAPs activate GTP hydrolysis, the consequences of myr 5 overexpression in living cells, and its subcellular localization. The myr 5 GAP activity exhibits a high specificity for Rho. To achieve similar rates of GTPase activation for RhoA, Cdc42Hs, and Rac1, a 100-fold or 1000-fold higher concentration of recombinant myr 5 GAP domain was needed for Cdc42Hs or Rac1, respectively, as compared with RhoA. Cell lysates from Sf9 insect cells infected with recombinant baculovirus encoding myr 5 exhibited increased GAP activity for RhoA but not for Cdc42Hs or Rac1. Analysis of Rho-family GAP domain sequences for conserved arginine residues that might contribute to accelerate GTP hydrolysis revealed a single conserved arginine residue. Mutation of the corresponding arginine residue in the myr 5 GAP domain to a methionine (M1695) virtually abolished Rho-GAP activity. Expression of myr 5 in Sf9 insect cells induced the formation of numerous long thin processes containing occasional varicosities. Such morphological changes were dependent on the myr 5 Rho-GAP activity, because they were induced by expressing the myr 5 tail or just the myr 5 Rho-GAP domain but not by expressing the myr 5 myosin domain. Expression of myr 5 in mammalian normal rat kidney (NRK) or HtTA-1 HeLa cells induced a loss of actin stress fibers and focal contacts with concomitant morphological changes and rounding up of the cells. Similar morphological changes were observed in HtTA-1 HeLa cells expressing just the myr 5 Rho-GAP domain but not in cells expressing myr 5 M1695. These morphological changes induced by myr 5 were inhibited by coexpression of RhoV14, which is defective in GTP hydrolysis, but not by RhoI117. myr 5 was localized in dynamic regions of the cell periphery, in the perinuclear region in the Golgi area, along stress fibers, and in the cytosol. These results demonstrate that myr 5 has in vitro and in vivo Rho-GAP activity. No evidence for a Rho effector function of the myr 5 myosin domain was obtained.