The structure of self-assemblies of amphiphiles formed at the air–aqueous solution interface can be determined by cryo transmission electron microscopy (Cryo-TEM). The method is based on fast vitrification of the thin layer of aqueous solution covered with amphiphilic monolayer by plunging the specimen into liquid ethane at its freezing point. During the process of fast cooling the aggregates maintain their two-dimensional crystalline integrity and structure, as demonstrated by comparative studies involving grazing incidence X-ray diffraction (GID) at the air-solution interface, and the Cryo-TEM measurements of the same amphiphilic systems on vitreous ice. Bright-field and dark-field images from the Cryo-TEM observations provide more detailed information than from epifluorescence and Brewster angle microscopy. Furthermore, the electron diffraction patterns have the additional advantage that they allow structural characterization of the crystallites almost at a molecular level, and furnish data on micro twinning and defects occurring between crystalline domains. Cryo-TEM has been applied to elucidate the structure of 2-D and 3-D self-aggregates of amphiphilic alcohols, acids and their cadmium salts, bola-amphiphiles and mixed monolayers. Epitaxial crystallization of hexagonal ice underneath the monolayer of long chain alcohol was also directly demonstrated by this method.