CD40 stimulation can synergize with interleukin (IL)-2 for antitumor responses against mouse metastatic renal cell carcinomas, with coincident increases in tumor-specific CD8+ T-cell responses and dendritic cell numbers in both the spleen and liver. Because CD40 is present on various hematopoietic-derived cells, endothelial cells, and some tumors themselves, this study was performed to determine whether the antitumor effects of CD40 stimulation and IL-2 were primarily mediated by CD40+ hematopoietic-derived cells. Bone marrow chimeras were created by reconstituting lethally irradiated CD40+/+ recipients with bone marrow from CD40-/- or CD40+/+ mice. Chimeric mice were then implanted orthotopically with renal cancer cells, followed by treatment with anti-CD40 agonist monoclonal antibodies and IL-2. Immune parameters of the spleen and liver were assessed after therapy and correlated with antitumor responses. The antitumor effects in the CD40-/- bone marrow transplantation chimeras were almost completely abrogated after treatment, and this shows that hematopoietically derived CD40+ cells are the principal targets for CD40 stimulation in this model. Although both spleen and liver showed reductions in CD8+ T-cell and dendritic cell expansion in the CD40-/- versus CD40+/+ chimeras after therapy, only the liver exhibited no significant increases in either CD8+ T cells or dendritic cells after treatment. CD40 cells on hematopoietic cells are the primary target for anti-CD40 and IL-2 therapy. The results also suggest that the immunologic events in the liver may be more revealing that those in lymphoid organs with regard to critical events related to responses after therapy.