A new method for flow cytometric analysis of calcium mobilization in human peripheral blood basophils without prior purification was developed. The method is based on dual color analysis of centrifugation-enriched mononuclear cell populations using fluo-3 and phycoerythrin (PE)-conjugated CD2, CD14, CD16, CD19 monoclonal antibodies (mAb) to stain contaminated cells. This technique allows the detection of fluo-3 fluorescence as a measure of an increase in the cytoplasmic free calcium concentration ([Ca(2+)]i) while simulataneously discriminating PE-mAb-unlabelled basophils. To clarify whether the human peripheral blood basophil is activated through the low affinity IgG receptor, Fc γ RⅡ, as well as the high affinity IgE receptor, Fc ε RⅠ, calcium mobilization after Fc γ RⅡ stimulation was analyzed by this method. After cross-linking of Fc γ RⅡ, transient [Ca(2+)]i elevation was observed but there was no apparent difference with interleukin-3(IL-3)-treated cells, and no significant histamine release was observed with or without short pre-incubation of IL-3. These findings suggest that the cross-linking of Fc γ RⅡ, not only Fc ε RⅠ, can activate human basophils which may result in mediator release other than histamine.