Natural killer (NK) cells are directly cytotoxic for tumor cells and play a primary role in regulating immune responses. We monitored levels of NK cell cytotoxic activity in cancer patients receiving D-Fraction extracted from maitake mushrooms (Grifola frondosa). Elevated levels of cytotoxic activity were maintained for one year. To elucidate the mechanisms underlying long-term activation of NK cells during treatment with D-Fraction, we examined tumor volume and levels of IFN-gamma and TNF-alpha in MM46-bearing C3H/HeN mice to which D-Fraction was administered for 19 d. D-Fraction markedly suppressed tumor growth, corresponding with increases in TNF-alpha and IFN-gamma released from spleen cells and a significant increase in TNF-alpha expressed in NK cells. This suggests that the D-Fraction activates NK cells even on the 20th day after treatment. Furthermore, D-Fraction increased macrophage-derived interleukin (IL)-12, which serves to activate NK cells. These results suggest that NK cells are not only responsible for the early effects of D-Fraction on tumor growth, but also for the long-term tumor-suppressive effects of D-Fraction through increased IL-12 released from macrophages.