Natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) eliminate virally infected and transformed cells. Target cell killing is mediated by the regulated exocytosis of secretory lysosomes, which deliver perforin and pro-apoptotic granzymes to the infected or transfomed cell. Yet despite the central role that secretory lysosome exocytosis plays in the immune response to viruses and tumours, little is known about the molecular machinery that regulates the docking and fusion of this organelle with the plasma membrane. In order to identify potential components of this exocytic machinery we used proteomics to define the protein composition of the NK cell secretory lysosome membrane. Secretory lysosomes were isolated from the NK cell line YTS by subcellular fractionation, integral membrane proteins and membrane-associated proteins were enriched using Triton X-114, separated by SDS-PAGE and tryptic peptides identified by LC ESI-MS/MS. In total 222 proteins were identified unambiguously in the secretory lysosome membrane fraction, of which 61% were predicted to be either integral membrane proteins or membrane-associated proteins. A significant proportion of the proteins identified play a role in vesicular trafficking, including members of both the Rab GTPase and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and protein families. These proteins include the Rab27a and the SNARE vesicle associated membrane protein-7 (VAMP7), both of which were enriched in the secretory lysosome fraction and represent potential components of the machinery that regulates the exocytosis of this organelle in NK cells.