The capability for sensitive and accurate identification of microorganisms has potential applications that include the monitoring of industrial bioprocessing operations, food safety analyses, disease diagnosis, and detection of potential biological hazards. Efforts based upon matrix-assisted laser desorption/ionization mass spectrometry to detect and identify specific microorganisms have been actively pursued for several years. We report a new method being developed to select useful biomarkers for the identification of microorganisms based upon electrospray ionization (ESI)-ion trap mass spectrometry. Crude cell lysates are processed using a recently developed dualmicrodialysis device and then directly infused into an ion trap MS. The low ESI flow rate and precursor ion accumulation capability of the ion trap MS enables high-sensitivity MS/MS analyses. Precursor ions are automatically selected and analyzed using tandem MS (MS/MS) to produce "global" MS/MS surveys and processed to yield two-dimensional MS/MS spectral displays. Such global MS/MS surveys are demonstrated for Escherichia coli lysates. The distinctive MS/MS spectral patterns can be used to identify mass spectrometric-detected species useful as biomarkers, which then provide a basis for confident microorganism identification. The results presented demonstrate the application of this method for the identification of microorganisms, as well as for detection of bacteriophage MS2 in the presence of a large excess of E. coli.