The immunomodulator ammonium trichloro (dioxyethylene-O-O')tellurate (AS101) has previously been found to induce secretion of various cytokines in mouse and human, which include interleukin-l, interleukin-2, colony-stimulating factor, interferon-gamma, tumor necrosis factor, etc. It also protects mice from lethal and sublethal effects of chemotherapy and irradiation. The present studies were designed to evaluate its effect on pulmonary metastasis following intravenous (i.v.) injection of mouse B16 melanoma cells on day 0 of the experiment. AS101, given 10 mu g/mouse intraperitoneally in 7 daily injections starting the day before B16 cell infusion (day -1) led to a significant inhibition by 60%. When B16 cells were pretreated with AS101 for 24 h before injection, the lung metastases were further reduced by subsequent AS101 treatment of the tumor-loaded mice. In mice that had been depleted of natural killer (NK) cells using anti-asialo-GM1 antisera, AS101 was deprived of its stimulatory effect on the NK activity. The inhibition by AS101 on the B16 lung metastasis was also profoundly reduced by the antisera. Moreover, in vitro treatment of B16 cells with AS101 resulted in suppression of the cell growth in a semisolid culture. This was accompanied by an inhibition of the DNA synthesis and a dephosphorylation/activation of the retinoblastoma susceptibility protein (RE), a tumor suppressor gene product, in the B16 cells. Taken together, these data suggest that AS101 possesses an anti-metastatic activity, which probably involves two mechanisms: the stimulation of the host NK cell activity and the inhibition of the tumor cell proliferation.