Granzyme A, a granule-associated serine proteinase of activated cytotoxic T cells and natural killer cells, has been reported to play a critical role in DNA fragmentation of target cells. To address the question of the biological role of granzyme A, we have now generated a granzyme A-deficient mouse mutant by homologous recombination. Western blot analysis, enzyme assays and reverse transcription-PCR confirmed the absence of granzyme A in activated T cells. In addition, deletion of granzyme A does not alter the expression patterns of other granule components, such as granzymes B-G and perforin. Granzyme A-deficient mice are healthy and show normal hematopoietic development. Most notably, their in vitro- and ex vivo-derived cytotoxic T cells and natural killer cells are indistinguishable from those of normal mice in causing membrane disruption, apoptosis and DNA fragmentation in target cells. Furthermore, granzyme A-deficient mice readily recover from both lymphocytic choriomeningitis virus and Listeria monocytogenes infections and eradicate syngeneic tumors with kinetics similar to the wild-type strain. These results demonstrate that granzyme A does not play a primary role in cell-mediated cytotoxicity, as has been assumed previously.