We have examined the ability of several serine proteases (granzymes) isolated from the granules of the rat natural killer cell line, RNK, to generate antigenic peptides of ovalbumin (Ova) that are capable of being recognized by Ova-specific CD8+ T cells. The mouse MHC class I-restricted cytotoxic T-cell clone, GX-1, which recognizes a trypsinized fragment of Ova in the context of H-2b, was able to lyse EL4 (H-2b) target cells in the presence of Ova and the granzymes but not in the presence of Ova or granzymes alone. Similar results were obtained using the murine Ova-specific CD8+ T cell hybridomas, RF33.70 and CD8OVA. In all cases, the T cells' responses were MHC class I-restricted as Ova:granzyme mixtures failed to mediate the lysis of the MHC-disparate target cell, P815 (H-2d). The purified rat granzyme preparations contained three distinct enzymatic specificities: asp-ase met-ase, and tryptase. Aprotinin, a protease inhibitor that only inhibits tryptase activity in vitro, completely abolished the CD8+ T-cell responses to Ova. These results, along with peptide loading studies using the RMA-S cell line, suggest that the granzyme treatment of Ova can generate the proper antigenic fragments which facilitate class I-restricted CTL responses both in vivo and in vitro. We believe that enzymes produced and released by NK or cytotoxic T cells within a tissue microenvironment may enhance the cleavage of target cell antigens as well as soluble antigens resulting in the improved uptake and processing of soluble antigens.