A method was established for measuring membrane fluidity of polymorphonuclear leukocytes (PMN) by the excimer-forming lipid technique with pyrenedecanoic acid in flow cytometry. When cells were labeled, the use of 2-25 microM of pyrenedecanoic acid provided similar results. Neither the removal of the unincorporated pyrenedecanoic acid nor adjustment of PMN counts exhibited any effect. By the gate analysis method, membrane fluidity of PMN could be measured with 100 microliters of heparinized whole blood in a short time and results with PMN in whole blood was similar to those with purified PMN. Therefore, purification and count adjustment of PMN could be omitted. By this method, membrane fluidity of PMN, which were treated with membrane fluidizer, was measured successfully. This method could be applied to the study of PMN function in various diseases.