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Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are able to lyse suitable target cells. CTLs recognize a specific peptide epitope presented by major histocompatibility complex (MHC) class I molecules on the target cells, whereas NK cells lyse targets that express no or low MHC class I molecules. Using perforin-deficient mice, we provide evidence that both NK cells and CTLs exclusively use perforin-dependent cytolysis as an effector mechanism in vitro, as well as in vivo. This review summarizes the most important role of perforin-dependent cytolysis in a wide variety of bacterial and viral infections, in tumour surveillance, in immunopathology and in autoimmunity.