Microphase-separated structure in cast triblock copolypeptide membranes was examined by fluorescence spectroscopy and electron microscopy. One of these membranes was composed of y-benzyl-L-glutamate and L-leucine (PBLG(x)-PLL(y)-PBLG(x)), (x = 0.18, y = 0.64, mole fraction), and the other was composed of L-glutamic acid and L-leucine (PLGA(x)-PLL(y)-PLGA(x)), which was prepared via saponification of the benzylglutamate groups of the PBLG-PLL-PBLG membrane. Electron micrograph of an ultrathin section of PBLG-PLL-PBLG membrane (cut perpendicular to the surface) showed that domains composed of the poly(y-benzyl-L-glutamate) chains were embedded in a continuous matrix of the poly(L-leucine) phase. The shape of the domains was nearly cylindrical, and the domain size (diameter) and the distance between two adjacent domains were estimated to be 100 and 140 nm, respectively. This domain structure was retained after the saponification reaction following which the domains become hydrophilic. Fluorescence emission spectra of 8-anilino-1-naphthalenesulfonic acid ammonium salt incorporated into wet PLGA-PLL-PLGA membrane showed three peaks. This suggests that the membrane has three regions with different degrees of hydrophobicity, which are presumed to represent the PLGA, interfacial, and PLL regions, respectively.