We have measured the production of volatile organic compounds (VOCs) by selected ion flow tube mass spectrometry (SIFT-MS) in the headspaces of conventional BacT/ALERT blood culture bottles (Biomerieux, Durham, NC) artificially infected with 5 bacterial strains. Uninfected blood samples were inoculated with Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and Neisseria meningitidis. Growth and species identification were determined at 6 h by measuring a panel of 9 VOC products. Two species, E. coli and S. aureus, were also incubated in the presence of gentamicin or flucloxacillin, respectively, above or below their demonstrated MIC. The concentration-dependent antibiotic susceptibility of both strains was demonstrated by the inhibition of VOC production at 22 h (P < .05). These results suggest incorporating SIFT-MS detection of microbial VOCs as a sensitive method for bacterial detection, identification, and determination of antibiotic susceptibility in a conventional blood culture system.