A number of proteins have specific phosphoinositide binding domains which target them to pools of phosphoinositide in the cell. One such domain is the pleckstrin homology (PH) domain of phospholipase Cδ1 (PLCδ1) which binds both PIP₂ and inositol trisphosphate (IP₃), and targets PLC to the cell membrane. Tubby protein (Santagata et al 2001) contains a tubby domain that selectively binds PIP₂.We investigated the use of a fluorescentlytagged version of this domain to monitor changes in PIP₂ concentration in living cells and compared it to the PH domain of PLCδ1.Yellow fluorescent protein (YFP) was fused to the tubby domain at the N (tubby-nYFP) and C-terminus (tubby-cYFP) using standard cloning techniques. PH-PLCδ1-CFP was kindly provided by Tobias Meyer. HEK293 cells were transiently transfected with each of these fluorescent constructs along with the G -coupled M₃ receptor using lipid based methods. Cells were visualised 24-48 hours after transfection using a confocal microscope, and superfused with bath solution (pH 7.4) containing (mM) 130 NaCl, 5 KCl, 10 Hepes, 8 glucose, 2.6 CaCl₂ and 1.2 MgCl₂.Values are from at least 2 transfections. PH-CFP was found to be localized to the cell membrane, but perfusion with 10 µM carbachol caused an increase in cytoplasmic fluorescence and a loss of membrane localization within 3-4 minutes (n=14). This was reversible on washout of carbachol and could be repeated. Preincubation with 50 µM wortmannin for 10 minutes did not remove the translocation effect of application of carbachol (n=14) or its reversability. In cells where IP₃ phosphatase was transiently overexpressed we observed limited translocation to the cytoplasm (4 out of 10 cells) during application of carbachol. In experiments where we overexpressed IP₃ phosphatase and preincubated with wortmannin, we did not observe any translocation of PH-CFP to the cytoplasm during application of carbachol (n=10). We observed that both tubby cYFP and tubby nYFP were localized to the cell membrane, and perfusion of carbachol as before did not result in translocation to the cytoplasm in tubby cYFP (n=15), but we did see reversible translocation of tubby nYFP in 2 out of 7 cells. After 10 minutes preincubation with wortmannin and subsequent application of carbachol, we observed translocation from the membrane to the cytoplasm in 12 out of 15 cells for tubby cYFP, and translocation to the cytoplasm in 4 out of 7 cells for tubby nYFP. We conclude that whilst translocation of PH-CFP may report changes in either IP₃ or PIP₂ concentration, the tubby domain reports only significant changes in PIP₂ concentration and is therefore a useful tool.