One mechanism by which monoclonal antibodies help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity via CD16 on Natural Killer cells. NK cells can engage multiple target cells sequentially and secrete perforin to induce their cytolytic killing. To study how NK cell degranulation is affected upon sequential stimulation, we designed a series of experiments where NK cells were sequentially stimulated with the same ligand or different ligand during the final round of stimulations. Using microscopy to visualize degranulation from individual NK cells, we found that repeated activation via CD16 decreased the amount of perforin secreted that was restored upon subsequent activation via a different activating receptor, NKG2D or NKP30. Repeated stimulation via NKG2D or NKp30 also decreased perforin secretion but this was not rescued by stimulation via CD16. By using transfected NK cells that express a non-cleavable mutant of CD16 we revealed that CD16 shedding is the key to these different functional outcomes. NK cells were stimulated by non-modified anti-CD20 (IgG1), while afucosylation is known to increase the affinity of mAbs for CD16 on NK cells. Next, we examined how mAb afucosylation affects the dynamics of NK cell interactions with opsonised target cells. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonisation of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNÎ³ secretion. Afucosylated mAb also caused a greater loss of CD16 from NK cell surfaces that has recently been described to be crucial for efficient NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of cell-cell interactions in an aqueous 2D environment and a 3D matrix, revealed that anti-CD20-AF induced more rapid killing of opsonised target cells. In addition, NK cells detached more quickly from target cells opsonised with anti-CD20-AF, which promoted serial killing of multiple targets. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and this can be augmented with antibody afucosylation.