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.