Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniae cultivated in Hep-2 cells were S-35-labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two-dimensional gel electrophoresis. Excised protein spots were in-gel digested with trypsin and peptides were concentrated on reverse-phase chromatographic beads for identification analysis by matrix-assisted laser desorption/ionization-mass spectrometry. In the pH range from 3-11, 263 C. pneumoniae protein spots encoded from 167 genes were identified. These genes constitute 15% of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.