Flowcytometric identification of basophils is a prerequisite for measuring activation of basophils with IgE-dependent or IgE-independent stimuli. Aim of this study was to compare different marker combinations in a simultaneous multicolor flowcytometric measurement. Methods Ten patients with a grass pollen allergy and 3 controls were included in the study. Basophilic cells were gated by using anti-CCR3647, anti-IgE, anti-CRTH2, anti-CD203c und anti-CD3. Cells were activated by a monoclonal anti-FcεRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and the allergen extract Phleum pratense. The activation marker anti-CD63 was used. Results The highest relative number of basophils was found with anti-CCR3(+) cells, anti-IgE(+) and anti-IgE(+) /anti-CD203c(+) cells, the lowest with CRTH2(+) /CD203c(+) /CD3(-) cells. A very good and good concordance of CCR3(+) cells was seen with CCR3(+) /CD3(-) cells and CRTH2(+) /CD203c(+) /CD3(-) cells in all experiments. The contamination of the CCR3(+) population with CD3(+) cells and the contamination of the IgE(+) -population with CCR3(-) cells and CD203(-) cells were the lowest compared to all other marker combinations. Conclusions As the highest relative number of basophils was identified by anti-CD193 (CCR3) followed by the anti-IgE and anti-IgE/antiCD203c positive population in most cases, these markers can generally be recommended for identification of basophils. If a basophil population with very high purity is needed, anti-IgE should be chosen.