A cDNA clone that is closely related to the granule-associated serine proteases of cytolytic T lymphocytes (CTL), called granzymes A-F, was isolated from a CTL expression library. The encoded serine protease, granzyme G, shows 70%-89% nucleotide identities to the granzymes C-F and, like those, consists of 228 amino acids preceded by the short propeptide Glu-Glu and a 18 residue long signal peptide. Granzyme G was identified by amino-terminal sequence analysis as a correctly processed and sorted protein stored in lysosome-like granules. The phylogenetic history of the granzyme multigene family was reconstructed by two tree-making methods and by Southern blot analyses of human, rat, and mouse DNA. Our results indicate differences in the evolutionary pathway between these species. The murine granzymes C-G descended from a progenitor present at the time of mammalian radiation. Granzyme C branched off first after the primate-rodent split and was involved in a recombination event with granzyme B before the rat-mouse divergence. Granzymes D and E have diverged after the mouse-rat speciation. However, no experimental evidence for the existence of a granzyme C-D-E-F-G equivalent was found in humans, and loss of the ancestral gene in the primate lineage is discussed. In view of the species differences in the number of granzyme gene copies during recent evolution, we propose that the murine granzymes B-G play several distinct roles in CTL-mediated effector functions as a response to quite recent changes of the biochemical environment.