We studied the effectiveness of monoclonal anti-CD40 + cytosine-phosphate-guanosine-containing oligodeoxynucleotide 1826 (CpG-ODN) immunotherapy (IT) in mice treated with multidrug chemotherapy (CT) consisting of vincristine, cyclophosphamide and doxorubicin. Combining CT with IT led to synergistic anti-tumour effects in C57BL/6 mice with established B16 melanoma or 9464D neuroblastoma. CT suppressed the functions of T cells and natural killer (NK) cells, but primed naïve peritoneal macrophages (Mφ) to in vitro stimulation with lipopolysaccharide (LPS), resulting in augmented nitric oxide (NO) production. IT, given after CT, did not restore the responsiveness of T cells and NK cells, but further activated Mφ to secrete NO, interferon-γ (IFN-γ) and interleukin (IL)-12p40 and to suppress the proliferation of tumour cells in vitro. These functional changes were accompanied by immunophenotype alterations on Mφ, including the up-regulation of Gr-1. CD11b(+) F4/80(+) Mφ comprised the major population of B16 tumour-infiltrating leucocytes. CT + IT treatment up-regulated molecules associated with the M1 effector Mφ phenotype [CD40, CD80, CD86, major histocompatibility complex (MHC) class II, IFN-γ, tumour necrosis factor-α (TNF-α) and IL-12] and down-regulated molecules associated with the M2 inhibitory Mφ phenotype (IL-4Rα, B7-H1, IL-4 and IL-10) on the tumour-associated Mφ compared with untreated controls. Together, the results show that CT and anti-CD40 + CpG-ODN IT synergize in the induction of anti-tumour effects which are associated with the phenotypic repolarization of tumour-associated Mφ.