In this study we test the hypothesis that reactive oxygen metabolites are delivered from neutrophils to simultaneously both the cell surface and cytosol of opsonized YAC erythroleukemic target cells. Using 5′ (or 6′) carboxyl‐2′,7′‐dichlorodihy‐drofluorescein (H₂‐CDCF) diacetate as starting material, we synthesized its succinimidyl ester derivative. H₂‐CDCF‐conjugated IgG prepared from the succinimidyl ester derivative was used to opsonize targets. In vitro studies have shown that H₂‐CDCF becomes fluorescent upon exposure to reactive oxygen metabolites, including hydrogen peroxide. Using video intensified epifluorescence microscopy, we observed that reactive oxygen metabolites are deposited on tumor cell membranes during neutrophil‐mediated antibody‐dependent cellular cytotoxicity (ADCC). This deposition process is catalase sensitive. The role of reactive oxygen metabolites produced by neutrophils in triggering the oxidation of H₂‐CDCF is further supported by the observation that neutrophils from chronic granulomatous disease (CGD) patients did not affect target fluorescence. YAC tumor cells were also labeled with dihydrorhodamine 123 or dihydrotetramethylrosamine. The oxidized forms of these reagents were found within the cytoplasm of YAC cells. During ADCC normal neutrophils, but not neutrophils obtained from CGD patients, triggered the oxidation of dihydrorhodamine 123 and dihydrotetramethyl‐rosamine within tumor cells. Using two‐color automated epifluorescence micros‐copy, we could not detect temporal intermediates with fluorescence in only one compartment, i.e., either solely on the plasma membrane or in the cytoplasm. These observations suggest that reactive oxygen metabolites cross target membranes (<12) sec. These studies show that reactive oxygen metabolites are deposited both onto and into tumor cells during ADCC, wherein both compartments could become vulnerable to oxidant‐mediated damage. © 1993 Wiley‐Liss, Inc.