Abstract I. Introduction 172 II. Bacterial RNA and DNA 174 III. Recombinant Proteins 175 IV. Protein Identification 176 V. Proteomics 178 VI. Other Applications that Involve Cellular Isolates 180 VII. Whole Cells 180 VIII. Interpretation of Data from Whole Cells 187 IX. Identification of Proteins and Metabolites in Cells 188 X. Isolate‐Related Strain Differences in Human Pathogenic Bacteria 190 XI. Conclusion and Future Prospects 190 References 191 The development of MALDI‐TOF mass spectrometry methods for the characterization of bacteria is reviewed and discussed. The general use of MALDI for the characterization of large biomolecules led directly to obvious applications involving the analysis of isolated bacterial proteins. More surprising was the observation that MALDI‐TOF mass spectrometry could be applied directly to crude cellular fractions or cellular suspensions and that the resulting data from such complex mixtures could provide evidence for chemotaxonomic classification. Versatility and the rapidity of analysis led to the rapid development of a number of MALDI‐TOF methods involving bacteria. Examples of some of the applications covered in this review are the analysis of bacterial RNA and DNA, the detection of recombinant proteins, the characterization of targeted or unknown proteins, bacterial proteomics, the detection of virulence markers, and the very rapid characterization of bacteria at the genus, species, and strain level. The demonstrated capability of taxonomic classification at the strain level, using unprocessed cells, opens the possibility that MALDI‐TOF and similar mass spectrometry approaches may contribute significantly to fulfilling emerging needs for the development of near real‐time methods for the characterization of bacteria. © 2002 John Wiley & Sons, Inc., Mass Spec Rev 20:172–194, 2001