Protein kinase C ε (PKC ε ) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKC ε and RhoA was defined. Phosphopeptide mapping revealed that PKC ε phosphorylates RhoA at T127 and S188. Recombinant PKC ε bound to recombinant RhoA in the absence of ATP indicating that the association between PKC ε and RhoA does not require an active ATP-docked PKC ε conformation. Activation of PKC ε resulted in a dramatic coordinated translocation of PKC ε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKC ε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKC ε -RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKC ε is able to recruit RhoA to the cell membrane indicating that the association between PKC ε and RhoA is proximal to the active catalytic site and perhaps independent of a PKC ε -RhoA phosphorylation event. This work demonstrates, for the first time, that PKC ε phosphorylates and modulates the cell membrane translocation of RhoA.