Lipid monolayer is a half leaflet model for lipid bilayer, which forms the basis of biological membranes. Within a certain range of surface area per molecule of phospholipid monolayers at the air-water interface, where the compressibility was nearly infinite, two phases with different molecular packings were observable by fluorescence microscopy. Mixed-phase monolayers of L-1,2-dipalmitoyl-N-monomethyl-3-phosphatidylethanolamine [DP(Me)PEl or L-1,2-dipalmitoyl-N-dimethyl-3-phosphatidyl-ethanolamine [DP(Me),PEl were deposited on marker grids coated with Formvar films. The molecular organization in the dark and bright fluorescent areas on the grids was investigated by low dose, selected area electron diffraction. Sharp reflection arcs, at a spacing of 4.2A and arranged in a hexagon pattern, were detected from dark domains of both lipids. A diffuse reflection ring at a spacing of 4.6A was derived from the bright background areas. Diffraction patterns were obtained from neighboring areas along selected dark domains of both lipids. The orientations of diffraction patterns from areas along smooth and curving boundaries of DP(Me),PE domains were found to turn with the boundaries. In the branching domains of DP-(Me)PE, the orientations of diffraction patterns indicated that the branches were formed by twinning. Electron diffraction thus provides an unique way to sample the local molecular packing order and orientation within individual domains in phospholipid monolayers. Lipid monolayers, Lipid bilayers, Electron diffraction o 1994 Wiley-Liss, Inc.*