Oxidative stress induces apoptosis in many types of cells, including human neutrophils. Our objective was to determine whether reactive oxygen species (ROS) produced by activated neutrophils are associated with accelerated apoptosis. Exposing neutrophils to ionomycin or phorbol myristate acetate (PMA) induced intracellular H₂O₂ production and rapid onset of apoptosis, measured as condensed chromatin, cellular shrinkage, and DNA fragmentation. Neutrophils activated with formyl‐methionyl‐leucyl‐phenylalanine (fMLP) generated mainly extracellular H₂O₂ and did not undergo apoptosis. Exogenously added H₂O₂, together with the catalase blocker sodium azide, induced apoptosis to the same extent and with similar kinetics as PMA and ionomycin. Adenosine inhibited ionomycin‐induced intracellular H₂O₂ production and apoptosis. Neither PMA nor ionomycin caused apoptosis in dimethyl sulfoxide‐differentiated HL‐60 cells, which are incapable of intracellular H₂O₂ production, whereas H₂O₂ induced apoptosis more efficiently in these cells than in neutrophils. We propose that activated neutrophils use intracellularly formed H₂O₂ to commit suicide. J. Leukoc. Biol. 65: 196–204; 1999.