An in vitro model for flow cytometric detection of heterogeneous drug response in exponentially growing Escherichia coli and Klebsiella pneumoniae was studied to evaluate the potential of this technology for rapid antibiotic susceptibility testing in polymicrobial samples. The cells, which exhibited 80-fold difference in in ampicillin susceptibility, were cultivated in the presence of ampicillin at a concentration equaling 1 MIC of the low-susceptibility strain (E. coli). Prior to flow cytometric analysis, the cells were fixed in 70% ethanol and stained with a DNA-specific dye. After 1 h of ampicillin incubation, the light scattering and fluorescence intensities of the susceptible cells increased 4.3-fold and 5-fold, respectively, but remained about constant for the resistant cells. The corresponding cell number increase for the resistant and the susceptible cells was 9.4 and 1.7, respectively. The two strains could be distinguished in the histograms on the basis of their light scattering and fluorescence intensities and by cell number. With an incubation of up to 3 h, the light scattering and fluorescence intensities of the susceptible cells grew stronger, at the expense of cell number. By combination of histograms, the discrimination of different cell populations could be improved. The results demonstrate the ability of flow cytometry to discriminate between species in heterogeneous cultures and suggest the potential of the technique for rapid assessment of bacterial susceptibility. However, the present results are preliminary, and the application to biological liquids and clinical samples has to be demonstrated further.