To determine the binding site of volatile anesthetics on purple membrane, diiodomethane (CH2I2) was used as a label in X-ray diffraction experiments. At less than 3 mM diiodomethane, purple membrane retains its two-dimensional crystallinity of bacteriorhodopsin, absorption spectra show only a little blue shift and the decay time of the M-intermediate becomes fast in flash photolysis experiments. These effects are similar to those of other anesthetics previously studied. The position where the anesthetic binds was identified by difference Fourier methods, and refined by model calculations. This study suggests that volatile anesthetics bind specifically to the protein-lipid interfacial region near the surface of membrane.