Direct injection electrospray ionization mass spectrometry (ESI-MS) without prior analyte separation was investigated for the analysis of whole cell suspensions of bacteria. Thirty-six strains of aerobic endospore-forming bacteria, consisting of six Bacillus species and one Brevibacillus species, were studied. ESI was performed in the positive ion mode on the bacterial suspensions. Several peaks in the range of 250-1500 m/z were observed to contribute to variations in the spectral information among the species. Application of cluster analysis to the spectral data showed that this ESI-MS technique was capable of discriminating strains of the species B. subtilis. This investigation demonstrates the feasibility of measuring liquid samples with minimal sample preparation that can be useful for discrimination at the subspecies level. A change in the cone potential in the electrospray ion source was found to influence the spectral information of representative strains of all of the seven species tested. This has implications with respect to optimizing the experimental conditions for discriminatory purposes, but could offer additional information with respect to microbial characterization. A comparison of the spectra of whole cell suspensions and cell-free supernatants showed a high degree of similarity between the two, which has consequences for ease of automation.