Gαi3 is found both on the plasma membrane and on Golgi membranes. Calnuc, an EF hand protein, binds both Gαi3 and Ca 2+ and is found both in the Golgi lumen and in the cytoplasm. To investigate whether Gαi3 binds calnuc in living cells and where this interaction takes place we performed fluorescence resonance energy transfer (FRET) analysis between Gαi3 and calnuc in COS-7 cells expressing Gαi3-yellow fluorescent protein (YFP) and calnuc-cyan fluorescent protein (CFP). The tagged proteins have the same localization as the endogenous, nontagged proteins. When Gαi3-YFP and calnuc-CFP are coexpressed, a FRET signal is detected in the Golgi region, but no FRET signal is detected on the plasma membrane. FRET is also seen within the Golgi region when Gαi3 is coexpressed with cytosolic calnuc(ΔN2–25)-CFP lacking its signal sequence. No FRET signal is detected when Gαi3(ΔC12)-YFP lacking the calnuc-binding region is coexpressed with calnuc-CFP or when Gαi3-YFP and calnuc(ΔEF-1,2)-CFP, which is unable to bind Gαi3, are coexpressed. Gαi3(G2AC3A)-YFP lacking its lipid anchors is localized in the cytoplasm, and no FRET signal is detected when it is coexpressed with wild-type calnuc-CFP. These results indicate that cytosolic calnuc binds to Gαi3 on Golgi membranes in living cells and that Gαi3 must be anchored to the cytosolic surface of Golgi membranes via lipid anchors for the interaction to occur. Calnuc has the properties of a Ca 2+ sensor protein capable of binding to and potentially regulating interactions of Gαi3 on Golgi membranes.