Granzymes are serine proteases released from the granules of cytotoxic lymphocytes during the induction of apoptosis. To evaluate the physiologic role of human granzyme K (GzmK), we developed a sensitive ELISA which was shown to specifically detect human GzmK in its active as well as its inactive conformation. Analysis of the lysate of lymphokine-activated killer (LAK) cells by gel filtration revealed that GzmK seems to be complexed to proteoglycans within these cells. While the expression of GzmA and B by cytotoxic lymphocytes was strongly up-regulated in response to several activating stimuli, GzmK expression did not increase significantly above constitutive levels, indicating differential regulation of these granzymes. However, low levels of GzmK were detected in plasma samples of healthy volunteers, which were in the same range as levels of GzmA and B. Furthermore, circulating levels of GzmK as well as of GzmA and B were significantly elevated in patients suffering from viral infections. We conclude that GzmK protein is produced by cytotoxic cells, and just as GzmA and B it can be released in a soluble form into the extracellular space. Furthermore, our data suggest that despite a more restricted cellular expression pattern, GzmK seems to participate in immune responses against several viruses.