Killer cell Ig-like receptors (KIRs) are MHC class I-specific receptors expressed in NK and T lymphocytes. KIR antagonism of activation signals occurs at the immune synapse between the effector and target cells. The processes that regulate clustering of KIR are not well defined. We have expressed KIR-GFP receptor chimeras in two human NK-like lines, YTS and NK92. In this study, we show that the frequency of KIR enrichment at the synapse was decreased for a KIR that lacks a portion of the cytoplasmic tail. Strikingly, blocking actin polymerization with a high dose of cytochalasin D also substantially decreased clustering of KIR as well as KIR-induced clustering of HLA-C-GFP in target cells. However, the effect of inhibiting actin polymerization was only clearly evident at the earlier time points after cell mixing, and eventually clustering of KIR and HLA-C occurred independently of actin remodeling. Although treatment with anti-LFA-1 also decreased conjugate formation, the frequency of KIR clustering remained normal within the population of conjugates that did form, suggesting that the effect of cytochalasin D is not solely through LFA-1. Collectively, these data suggest that the actin cytoskeleton and the cytoplasmic tail of KIR regulate the efficiency by which KIR accumulates at inhibitory NK cell synapses.