Antibiotic-resistant strains of bacteria continue to emerge, increasing the need for their fast and accurate identification. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), has become a prominent technique in biological mass spectrometry. We report the application of MALDI-TOF-MS for the identification of intact Gram-negative and Gram-positive microorganisms taken directly from culture. Analysis of bacteria from a single colony is possible, allowing the screening of mixed cultures. Sample preparation is simple and the analysis automated, providing spectra within minutes. The spectra obtained allow identification of microorganisms from different genera, different species, and from different strains of the same species. The procedure provides a unique mass spectral fingerprint of the microorganism, produced from desorbed components of the cell wall. Consistent data were obtained from subcultures grown for 3-day and 6-day periods, from the same cultures 1 day later and from fresh subcultures 2 months later.