Natural Killer (NK) cells interact with other cells through a structured interface, the immune synapse (IS). A balance of signals controls NK cell activity through ligation of activating and inhibitory receptors. If signaling favours activation, NK cells mediate the directed secretion of cytotoxic mediators, such as perforin (PFN). To test whether the IS also functions as a gasket to extracellular molecules, fluorescently labeled nanometer-scale dextrans of varying sizes were co-incubated with effector-target cell-cell conjugates. Quantitative fluorescence microscopy of synapses revealed that dextrans with hydrodynamic diameters ≥32 nm were excluded from activating synapses, whilst smaller dextrans could enter. Size-dependent exclusion required an intact filamentous actin scaffold, but not continuous reorganisation following synapse formation. Time-lapse microscopy further revealed that the synapse assembles in a zipper-like manner, clearing larger dextran from the synapse. In addition, monoclonal antibodies and low-density lipoproteins were also excluded from the IS, whereas smaller domain antibodies could penetrate. NK cells can lyse more than one target in series. Whilst it is known that, among other proteins, PFN is secreted to lyse diseased cells, the amount of PFN secreted by NK cells is currently unknown. To quantify PFN release following stimulation through NKG2D or CD16, NK cells were plated on protein-coated surfaces that could capture PFN. Quantification using fluorescence microscopy revealed that PFN secretion was analogue, varying with increased ligand density. Simulating serial killing, repeated stimulation decreased the amount of PFN secreted with sequential activation. Unexpectedly, CD16 stimulation following serial NKG2D ligation recovered this decrease in secretion, however a similar recovery was not seen under reciprocal conditions. These data show that the activating IS clears and excludes extracellular molecules, including antibodies, in a size-dependent manner. Further, NK cell PFN secretion is an analogue response that varies with both ligand density and the receptors ligated in series.