Although the styryl dye FM4–64 is now used routinely to monitor endocytosis in plants, the argument about its potential to cytoplasmically and non-endocytically relocate into a selective set of vesicular compartments persists. To address this question, we determined whether fluorescence resonance energy transfer (FRET) could occur between a cytoplasmically expressed, short-wavelength excitation green fluorescent protein (GFP) and FM4–64 in Nicotiana benthaminana. After exposure to FM4–64, the root hair plasma membrane and internal organelles became labelled. Under these conditions, no FRET with cytoplasmic GFP was seen. However, if the cells were treated with a low concentration of quillajasaponin, a membrane permeabilization agent, the cells continued to stream and FRET was detected. Thereby, we demonstrate that under conditions that do not severely compromise cell viability, the FM4–64 dye becomes a suitable FRET partner for the cytoplasmically localized GFP. Under normal conditions, FM4–64 does not significantly enter the cytosolic side of the membrane, but remains at the plasma membrane or trapped in the organelles of the endocytic pathway. Hence, when the structure or permeability of the plasma membrane is unaltered, FM4–64 dye is a robust marker for endocytosis.