Many cellular processes are regulated by phosphoinositides (PI) in the cell membrane. Studying these regulatory mechanisms has been greatly advanced by the development of genetically encoded fluorescent probes that can be used to dynamically monitor PI concentrations ([PI]). However, genetically encoded tools that allow to efficiently manipulate [PI] remain scarce. One such tool is the voltage sensitive PI(3,4,5)P₃/PI(4,5)P₂-5'- phosphatase Ci-VSP, which converts PI(3,4,5)P₃ to PI(3,4)P₂ and PI(4,5)P₂ to PI(4)P. The catalytic activity of Ci-VSP is finely controlled by membrane potential (V_m) via a Voltage Sensing Domain in the N-terminus. This important feature grants temporal control over the catalytic domain. The C-terminus of Ci-VSP constitutes the phosphatase domain (PD), which shares high homology with the PI(3,4)P₂/PI(3,4,5)P₃-3'- phosphatase PTEN. We replaced the PD of Ci-VSP by PTEN and coexpressed the resulting construct, termed Ci-VSPTEN, with GFP tagged PI(3,4,5)P₃ sensor domains in CHO cells. Depolarization of the cell from the resting V_m of -60 mV to 80 mV led to a strong translocation of the PI(3,4,5)P₃ sensors from the membrane into the cytosol, as detected by TIRF microscopy. In contrast, PI(4,5)P₂ sensing domains showed no such translocation when co-expressed with Ci-VSPTEN. These results indicate voltage dependent PI(3,4,5)P₃-, but not PI(4,5)P₂-phosphatase activity of Ci- VSPTEN. In light of the known 3'-phosphatase activity of PTEN we conclude that the chimera targets the 3' position. Thus Ci- VSPTEN extends the toolset available for [PI] manipulation by a voltage sensitive PI phosphatase with novel specificity. In this work voltage sensitivity was conferred to a cytoplasmic enzyme. Similar approaches might be used to develop "designed" voltage sensitive enzymes, allowing the interference with membrane-dependent processes. Supported by DFG grant OL240/2 & SFB593 to DO and NIH grant GM30376 to FB