The localization of antigens and other substances in cells and tissues by electron microscopy is usually performed by immunohistochemical techniques employing labelled conventional or monoclonal antibodies. For the ultrastructural localization of the antibodies, they are coupled to electron-dense labels like gold or ferritin. Here, we demonstrate a novel method to localize antigens in cells, tissues, and on other supports. By electron energy loss spectroscopy (EELS) it is possible to directly analyze the distribution of antigens, metabolites or other substances without the use of labelled antibodies: as an example we demonstrate the distribution of the immunomodulator lipopeptide in B lymphocytes and macrophages. EELS represents a novel, sensitive, and generally applicable method for the detection and localization of antigens and other substances in biology and medicine.