Human neutrophils aggregate after stimulation with various stimuli; this response is completely absent in neutrophils from patients with leukocyte adhesion deficiency (LAD). To investigate the cellular requirements of this process a method was used in which neutrophils are separately loaded with hydroethidine (HE) and sulfofluorescein (SFDA), to give them either red fluorescence or green fluorescence. After mixing HE- and SFDA-labeled cells in a ratio of 1:1, the number of double-colored aggregates formed after activation was determined by analysis on a fluorescence-activated cell sorter. In this way, essential information is obtained when cells of different origin are used. The formation of aggregates between neutrophils of an LAD patient and control neutrophils was thus quantified. Because neutrophil aggregation is dependent mostly on the presence of complement receptor type 3 (CR3), which is not present on LAD neutrophils, this result revealed the presence of a counter-structure for CR3 on LAD neutrophils (and hence on normal human neutrophils). In addition to the presence of these proteins on the cell surface, aggregation required continuous cell triggering as indicated by the transient aggregation induced by short-term activation of protein kinase C. This phenomenon was substantiated by the fact that energy depletion caused profound disaggregation. The present study reveals that neutrophil aggregation is a well-controlled process, which needs constant activation of CR3 for a stable interaction with a constitutively expressed counter-structure.