AgrC is an integral membrane receptor protein with histidine kinase activity in the accessory gene regulator (agr) quorum-sensing system of Staphylococcus aureus. In this study, proteoliposomes were used as a model to investigate AgrC orientation. Many approaches have been described to determine membrane protein orientation, but they are often complicated and time consuming. In this study, AgrC orientation in liposomes was determined by thiol-reactive reagent labeling and a kinase activity assay. Our results suggest use of a kinase activity assay could get an accurate percentage of functional protein orientation and only cost nearly one-sixth of the time compared with the method based on thiol-reactive reagent labeling. We present an effective and rapid method for determining the orientation of membrane protein kinases like AgrC. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.