The cytolytic potential of T effector cells appears to be intimately related to the presence of proteins stored in specialized cytoplasmic granules. A striking biological property of isolated granules is their lytic activity for a variety of target cells in a nonrestricted manner. Proteins contained within these granules of CTLs are specifically released upon target cell recognition. We have isolated and characterized six granule-associated proteins in two murine CTL lines in addition to the pore-forming and target membrane-disrupting perforin. Six full length cDNA clones have been identified in a CTL-specific cDNA expression library which code for the granule-associated serine esterases, designated as granzymes A to F. Granzymes A and B represent the genuine proteins encoded by the H factor/CTLA-3 cDNA and the CTLA-1/CCPI cDNA, respectively. The covalent amino acid structures of all six granzymes show the hallmarks for serine proteases and are highly related to that of rat mast cell protease I and II and cathepsin G, which have been found in granules of mast cells and neutrophilic granulocytes, respectively. The primary translation products are processed by removal of a hydrophobic signal peptide and a two residue-long propeptide at the amino-terminus. Immuno-electron microscopy shows that granzymes and perforin are stored together within secretory granules of CTLs. Simultaneous release of at least two of these granzymes has been observed during degranulation of a murine CTL line by anti-T3 antibodies. The biological role, particularly the proteolytic events elicited by granzyme A and other granzymes in the context of target cell recognition, are not known at present. It is unlikely that they form a proteolytic activation cascade together with pore-forming proteins analogous to the complement system. The strictly regulated secretion of granzymes and the lack of measurable enzymatic activity in the case of granzymes B, C, E and F towards a variety of synthetic substrates suggest a highly specific function for each of them.