Diphenylhexatrienylpropanoylhydrazyl stachyose (glyco-DPH), a new fluorescence probe, was synthesized. It inserts almost instantaneously into artificial phospholipid vesicles and biological membranes. Due to its large hydrophilic carbohydrate portion, it serves as an uncharged probe with a defined orientation within the membrane bilayer. Its usefulness to monitor lipid mobility by means of its fluorescence anisotropy could be demonstrated for dipalmitoylglycerophosphocholine at temperatures around the gel to liquid phase transition and the rigidifying effect of cholesterol on egg yolk phosphatidyicholine membranes. In addition, lipid mobility was determined for biological membrane systems such as yeast spheroplasts, yeast organelles, cultured human skin fibroblasts and compared with the 'fluidities’ of vesicles made of the corresponding lipid extracts. Bimodal Lorentzian lifetime distributions determined for glyco-DPH in vesicles of 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine showed that the label is distributed homogeneously within a phospholipid bilayer. Fluorescence microscopy of living (fibroblast) cells revealed selective labeling of the surface membrane with glyco-DPH under appropriate conditions.