Defects of planarity of two dimensional crystals constitute a major limitation in high-resolution structural studies of biological macromolecules by electron crystallography. We have found that reflected light microscopy constitutes a simple and appropriate technique for revealing the state of planarity of carbon films commonly used for supporting specimens in electron microscopy. Carbon films evaporated onto mica and deposited onto copper grids presented a highly wrinkled aspect. In contrast, carbon films prepared after evaporation on nitrocellulose presented large areas with an homogeneous flat aspect, while other areas were wrinkled. The wrinkled aspect was also detected by transmission electron microscopy at extremely high values of defocusing, by scanning electron microscopy using highly tilted specimens, and was emphasized by Nomarski differential interference contrast in reflected light microscopy. We further characterized the surface of carbon films by atomic force microscopy. The wrinkled aspect was shown to be directly related to the presence of folds extending across carbon films. Typical values for the lateral and vertical dimensions of the folds were several micrometers and several hundred nanometers, respectively. These defects of carbon film planarity correspond to a local tilting of carbon films of the order of 1 degrees. We believe that the wrinkled nature of carbon films is a general feature of crucial importance for high-resolution studies. (C) 1991 Academic Press, Inc.