The aim of this study was to establish a standard flow cytometric method to measure the phagocytic function of and intracellular hydrogen peroxide (H₂O₂) production by rat leukocytes. Thirty-six adult, male Sprague–Dawley rats were included in this study. Whole-blood specimens from the inferior vena cava were collected in a heparinized tube and ethylenediaminetetraacetic acid (EDTA) anticoagulated tube. The phagocytic function of and intracellular H₂O₂ generation by leukocytes were measured with FACS Vantage™ flow cytometer (Becton Dickinson, San Jose, CA), using fluorescent microspheres and dihydrorhodamine-123 as probes, respectively. Several conditions were optimized in this study, including anticoagulants (heparin and EDTA), fluorescent probes (0.75- and 1.72-μm-diameter microspheres), incubation time, and concentration of the chemicals used in the experiment. Neutrophils, monocytes, and lymphocytes could be clearly defined and separated in whole blood by flow cytometry and tested for phagocytosis and intracellular H₂O₂ generation without the need for further purification and handling of the cells. Intracellular H₂O₂ production by and phagocytic function of neutrophils and monocytes were inhibited in EDTA-anticoagulated blood compared with heparin- anticoagulated blood (P < 0.01). Neutrophils showed similar phagocytic function to 0.75- and 1.72-μm microspheres, but monocytes showed weak phagocytic activity to 1.72-μm beads compared with 0.75-μm beads (P < 0.01). In conclusion, a flow cytometric method to measure the phagocytic function of and intracellular H₂O₂ production by rat leukocytes has been developed. Quantitative flow cytometric analysis of rat leukocyte function is convenient and feasible and provides a reliable and rapid assay to assess phagocytosis and intracellular H₂O₂ production by rat neutrophils and monocytes.