Phosphatidylinositol 4,5-bisphosphate (PIP₂) has important regulatory roles for many cellular processes such as activity of ion channels, cytoskeleton remodelling and vesicle trafficking. The PIP₂ level itself can be regulated by many G_q/11-coupled receptors. Imaging of genetically encoded fluorescently tagged sensors based on the pleckstrin homology domain of PLCd1 (PHdom) or the C-terminal PIP₂ binding domain of tubby (tubby-CT) is a promising method to address spatiotemporal dynamics of PIP₂ in living cells. In this method, the degree of membrane association of PHdom or tubby-CT is a direct measure for the PIP₂ concentration within the membrane. During PLC activation via the muscarinic M1 receptor PHdom-GFP showed translocation from the membrane to the cytosol indicating PIP₂ depletion. However, although tubby-CT showed translocation in some cells, a substantial population of the cells showed no or only margnal tubby-CT responses. Moreover tubby-CT responses were highly variable, such that degrees of translocation of the both sensors in cotransfected cells were only loosely correlated. In contrast, we observed full translocation of both sensors in response to PIP₂ depletion induced by the voltage dependent 5'-phosphatase, Ci-VSP. The degree of translocation of both sensors induced by sequential activation of PLC and Ci-VSP in the same cell indicated that their differential behavior cannot be explained by distinct affinities to PIP₂. Combined activation of PLC and Ci-VSP in the same cell suggested that PHdom and tubby-CT may report on distinct PIP₂ pools with differential accessibility for PLC.