The cytoplasmic, dense granules of cloned T cell lines were isolated and analyzed for their functional and biochemical properties. Isolated granules of approximately 90% homogeneity, in the presence of Ca, effect strong tumoricidal and hemolytic activity. Tumor cell lysis is complete in less than 30 min, with less than 2 micrograms granule protein corresponding to a killer/target ratio of 3-6:1 by assuming 50% yield for granule isolation. The granules contain a set of unique proteins, responsible for cytolytic activity and designated K1 to K6, in the molecular weight range of 14,000 to 75,000, as defined by sodium dodecyl sulfate (SDS) polyacrylamide slab gel analysis under reducing and nonreducing conditions. Cytolysis mediated by isolated granules is accompanied by the assembly of tubular complexes of 160 A (poly P1) and of approximately 70 A width (poly P2) that are inserted into membranes and form ultrastructural membrane lesions. As shown by immunofluorescence and by Percoll gradient fractionation, cytolytic granules are detected in cells of cytolytic T cell lineage and not in the T cell lymphomas E14 and S194. Poly perforin 1 assembled by CTLL-2 upon stimulation with concanavalin A (Con A) and phorbol myristate acetate (PMA) was isolated by detergent extraction and gel filtration. Poly P1 is composed of disulfide-linked subunits that, after reduction, co-migrate with certain granule proteins. The results are compatible with the hypothesis that the dense granules of cytolytic T cells contain cytolytic proteins that polymerize to disulfide-linked tubular poly perforins in a Ca-dependent reaction and may cause cytolysis by membrane insertion and transmembrane channel formation.