Although blood neutrophils with toxic granulation provide an excellent means of evaluating acute bacterial infections, the methods are labor-intensive and their reproducibilities depend on the staining technique and the observer's judgment. We measured the flavin adenine dinucleotide (FAD) content of normal neutrophils and neutrophils with toxic granulation by flow cytometry after incubating them with bis(Zn2+-dipicolylamine) complex. A total of 122 blood samples (78 with neutrophils with toxic granulation, 44 with normal neutrophils without toxic granulation) were analyzed. The mean autofluorescence levels of neutrophils in the toxic granulation (+) group and toxic granulation (-) group were both 1.9 × 103 MESF (mean equivalent soluble fluorochrome) values. However, after incubating neutrophils with bis(Zn2+-dipicolylamine) complex for 15 min, the mean fluorescence intensities of neutrophils in the toxic granulation (+) and toxic granulation (-) groups were significantly enhanced by 71- and 19-fold to 138±78 × 103 MESF and 37±37 × 103 MESF, respectively (p<0.001). An MESF cutoff value of 79 × 103 showed a sensitivity of 93.2% and a specificity of 81.8% for neutrophils with toxic granulation. Similarly, the MESF level ratio, defined as the ratio of the MESF value after incubation with bis(Zn2+-dipicolylamine) complex to the autofluorescence MESF value, at a cutoff value of 41, showed a sensitivity of 92.3% and a specificity of 81.8% for the detection of neutrophils with toxic granulation. The MESF values of neutrophils after incubation with bis(Zn2+-dipicolylamine) complex did not correlate with the leukocyte (p >0.05) or neutrophil counts (p >0.05). In conclusion, measurement of FAD fluorescence intensity in neutrophils by flow cytometry after incubation with bis(Zn2+-dipicolylamine) complex is an easy, objective, and reliable method of detecting neutrophils with toxic granulation.