Granzymes are a family of serine proteases found in the lytic granules of cytotoxic T lymphocytes and natural killer (NK) cells, which are involved in killing of susceptible target cells. Most information on granzymes and their enzymatic specificities derive from studies in humans and mice. Although granzymes shared by both species show a high level of conservation, the complement of granzyme genes differs between the species. The aim of this study was to identify granzyme genes expressed in cattle, determine their genomic locations and analyse their sequences to predict likely functional specificities. Orthologues of the five granzyme genes found in humans (A, B, H, K and M) were identified, as well a novel gene designated granzyme O, most closely related to granzyme A. An orthologue of granzyme O was found in pigs and a non-function version was detected in the human genome. Use of specific PCRs demonstrated that all of these genes, including granzyme O, are expressed in activated subsets of bovine lymphocytes, with particularly high levels in CD8 T cells. Consistent with findings in humans and mice, the granzyme-encoding genes were located on three distinct genomic loci, which correspond to different proteolytic enzymatic activities, namely trypsin-like, chymotrypsin-like and metase-like. Analysis of amino acid sequences indicated that the granzyme proteins have broadly similar enzymatic specificities to their human and murine counterparts but indicated that granzyme B has a different secondary specificity. These findings provide the basis for further work to examine their role in the cytotoxic activity of bovine CD8 T cells.