AbstractA major difficulty in preparing frozen‐hydrated specimens from solutions is to control an optimal thickness of water remaining on the EM grid just before it is immersed in liquid nitrogen. In this report, we describe a modification of the fatty acid monolayer technique for preparing frozen‐hydrated specimens. With the use of this technique, all excess solvent is removed when a behenic acid (a fatty acid) monolayer film is picked up over the surface of the electron microscope grid. Low‐dose electron diffraction patterns and optical diffraction patterns of low‐dose micrographs of bacteriorhodopsin (bR) ofHalobacterium halobiumthus prepared show strong reflection orders at a resolution of ∽4 Å and ∽10 Å, respectively. Because the preparation of thin, hydrated specimens by the behenic acid monolayer technique does not depend upon evaporation of water, a dramatic change of ion concentration and pH is not expected to occur. The method should be applicable to specimens which need to be kept in a specific buffer and/or at a specific i