A single treatment of low-temperature, long-duration, whole-body hyperthermia of either severe combined immunodeficient (SCID) mice bearing human breast tumor xenografts or Balb/c mice bearing syngeneic tumors for 6-8 hr can cause a temporary reduction of tumor volume and/or a growth delay. In both animal model systems, this inhibition is correlated with the appearance of large numbers of apoptotic tumor cells. Because this type of mild heat exposure, comparable to a common fever, is not itself directly cytotoxic, other explanations for the observed tumor cell death were considered. Our data support the hypothesis that this hyperthermia protocol stimulates some component(s) of the immune response, which results in increased antitumor activity. In support of this hypothesis, increased numbers of lymphocyte-like cells, macrophages, and granulocytes are observed in the tumor vasculature and in the tumor stroma immediately following this mild hyperthermia exposure. In Balb/c mice, an infiltrate persists in the tumor for at least 2 weeks. Using the SCID mouse/human tumor system, we found that both host natural killer (NK) cells and injected human NK cells were increased at the site of tumor following hyperthermia treatment. Experiments using anti-asialo-GM1 antibodies indicate that the tumor cell apoptosis seen in the SCID mouse appears to be due largely to the activity of NK cells, although additional roles for other immunoeffector cells and cytokines appear likely in the immunologically complete Balb/c model. Another interrelated hypothesis is that immunoeffector cells may have greater access to the interior of the tumor because we have observed that this treatment causes an obvious expansion in the diameter of blood vessels within the tumor and an increase in nucleated blood cells within the vessels, which persists as long as 2 weeks after treatment. Further study of the mechanisms by which mild hyperthermia exerts antitumor activity could result in this treatment protocol being used as an effective, nontoxic adjuvant to immunotherapy and/or other cancer therapies.