Combined continuous s.c. coadministration of macrophage-colony stimulating factor (M-CSF) plus interleukin-2 (IL-2) by osmotic pump protected mice given i.v. injections of a lethal dose of EL4 T-cell leukemia/lymphoma. Antitumor protection was significantly greater than that afforded by treatment with either cytokine alone. Since neither IL-2 receptors nor M-CSF receptors were expressed on EL4, the antitumor effect was likely attributed to murine effector cells. To determine how M-CSF + IL-2 provided this effect, we performed immunophenotypic and functional analyses as well as in vivo depletion studies of putative antitumor effector cells. Splenic phenotyping experiments revealed that the highest levels of macrophages and natural killer cells were observed in mice given the cytokine combination rather than either M-CSF or IL-2 alone. In vivo depletion of natural killer cells ablated the antitumor protective effect of M-CSF and IL-2. T-cells were also important for M-CSF + IL-2 efficacy, since adult thymectomy/T-cell depletion significantly inhibited the ability of cytokine coadministration to protect against EL4. Coadministration of the 2 cytokines significantly elevated in vivo levels of CD3⁺CD4⁺, CD3⁺CD8⁺, CD3⁺NK1.1⁺ T-cells, and CD3⁺CD25⁺ (activated) T-cells, and elevated anti-EL4 cytotoxic T-cell activity measured in vitro. Although WBC counts and fluorescence-activated cell sorter studies showed that M-CSF + IL-2 treatment significantly elevated neutrophils, s.c. delivery of granulocyte-colony stimulating factor at doses sufficient to induce neutrophilia was unable to confer anti-EL4 protection. These studies indicate that macrophages, T-cells, and natural killer cells are all important in the M-CSF + IL-2 anti-EL4 response. The superior antitumor effect of this cytokine combination along with the ability of M-CSF to diminish the toxicity of IL-2 in this model suggests that further investigations into the clinical potential of this combination treatment are warranted.