Medullasin, a serine protease in bone marrow cells, has been shown to enhance human natural killer cell activity by acting directly on large granular lymphocytes. In the present report the mechanism of activation of natural killer cells by medullasin was studied by employing a single cell assay method in agar. Medullasin enhanced the target-binding capacity of lymphocytes, but the percentage of target-binding cells with dead target remained essentially unchanged. The maximum recycling capacity of natural killer cells was also increased by the treatment with medullasin. These results indicate that medullasin in granulocytes stimulates the maturation of natural killer cells with elevated recycling capacity from human large granular lymphocytes.