Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was investigated as a method for the rapid identification of whole bacteria, either by comparison with archived reference spectra or by co-analysis with cultures of known bacteria. Bacteria were sampled from colonies on an agar plate, mixed with the matrix, air-dried, and introduced in batches into the mass spectrometer for analysis. In the first experiment, both bacterial strains that had been previously analyzed to obtain reference spectra and other strains that had not been analyzed were blind-numbered and their spectra were obtained. Those strains that matched reference spectra were found to be correctly identified. A second experiment involved co-analysis of reference strains and bind-numbered strains under identical conditions; species-specific identification was demonstrated by comparison of spectra of the blind-numbered strains with those of the standards. In all of the spectra obtained in these experiments, each bacterial strain showed a few characteristic high-mass ions which are thought to be derived from bacterial proteins. This work represents the first reported instance of successful bacterial chemotaxonomy by MALDI-TOFMS analysis of whole cells. For the strains tested, the method is rapid and simple.