Cellular proteins (biomarkers) specific to any individual microorganism, determined by the direct mass spectral analysis of the corresponding intact cellular suspension, can be applied for the rapid and specific identification of the organisms present in unknown samples. The components of the bacterial suspensions, after a rapid separation over a C 18 reversed-phase microcapillary column, were directly subjected to on-line electrospray ionization followed by analysis using an ion trap tandem mass spectrometer. This approach is equally effective for gram-positive as well as gram-negative bacteria but has a distinct advantage over our earlier reported method involving matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). During electrospray ionitation mass spectrometry (ESI-MS), liquid samples can be directly analyzed and there is the potential for developing tandem mass spectral methods for more specific identification of the individual organisms present in crude bacterial mixtures. The total analysis time leading to unambiguous bacterial identification in samples was less than 10 minutes and the results were quite reproducible. Miniaturization of the instrumentation along with total automation of this simple process could have immense impact on field operations. Routine, rapid, cost-effective field monitoring of environmental samples, agricultural products, samples from food processing, industrial sites and health institutions for suspected bacterial contamination could be a reality in the near future. Potential utility in biological, medical, bioprocessing, pharmaceutical, and other industrial research is also enormous.