Natural killer (NK) cells and cytotoxic T-lymphocytes (CTL) deploy perforin (Prf) and granzymes to kill infected host cells. Perforin, secreted by immune cells, binds target membranes to form pores that deliver pro-apoptotic granzymes into the target cell. A crucial first step in this process is interaction of the C2 domain with target cell membranes, which is a calcium-dependent event. Some aspects of this process are understood, but many molecular details remain unclear. To address this, we investigated the mechanism of Ca2+ and lipid binding to the C2 domain by NMR spectroscopy and X-ray crystallography. Calcium titration, together with dodecylphosphocholine (DPC) micelle experiments confirmed that multiple Ca2+ ions bind within the calcium binding regions (CBR), activating perforin with respect to membrane binding. We have also examined the retaliative affinities of several of these binding sites and demonstrated that this interaction causes a significant structural rearrangement in CBR1. Thus, it is proposed that Ca2+-binding at the weakest affinity site triggers changes in the C2 domain that facilitate its interaction with lipid membranes.