We investigated the regulatory mechanisms controlling the subcellular localization of p21-activated kinase 5 (PAK5) and found that the Cdc42/Rac interactive binding (CRIB) domain within PAK5 is critical for proper targeting within the cell. We also observed that PAK5 interacts with RhoD and RhoH in addition to Cdc42, and that interaction with RhoD targets PAK5 to subcellular locations that are distinct from those stimulated by Cdc42. Through deletion analysis we observed that the mitochondrial localization of PAK5 is controlled by multiple domains, providing evidence that the kinase activity of PAK5 is critical to its ability to cycle on and off mitochondria, and demonstrate that expression of kinase-inactive PAK5 elicits dramatic effects on mitochondrial morphology. These data indicate that PAK5 is directed to distinct subcellular locations by different Rho family small G proteins as well as by intrinsic targeting sequences. (c) 2006 Elsevier Inc. All rights reserved.