AbstractWe investigated in detail the cation requirements of two different systems of mouse cell‐mediated cytolysisin vitro, at their recognition, post‐recognition hit and target cell disintegration stages. In T cell‐mediated cytolysis, respectively Mg++or Ca++, Ca++, and no cations, were required. In non‐T cell‐mediated hemolysis, respectively no cations, Mg++, and no cations, were required. Two main conclusions can be drawn. First, the cation requirements are different from one system to the other especially at the post‐recognition hit stage, which strongly suggests the existence of differences between both systems as to the actual mechanism of lysis. Second, the cation requirements are different within each system from one step to another. This formed the basis of a step‐by‐step analysis of the lytic process, leading to the characterization of a cation pulse method for the experimental isolation and further study of the post‐recognition hit stage of cell