Effector cells of the innate immune system have diverse functions that can result in tumour inhibition or tumour progression. Activation of macrophages by CD40 ligation has been shown to induce antitumour effects in vitro and in vivo. Here we investigated the role of nitric oxide (NO) and tumour necrosis factor-α (TNF-α) as mediators in the tumoristatic effects of murine peritoneal macrophages activated with agonistic anti-CD40 monoclonal antibody (αCD40) alone and following further stimulation with bacterial lipopolysaccharide (LPS). We found that macrophages activated in vivo by αCD40 exhibited tumoristatic activity in vitro against B16 melanoma cells; the tumoristatic effect correlated with the level of NO production and was enhanced by LPS. Use of the NO inhibitor l-nitro-arginine-methyl esterase (L-NAME) and evaluation of macrophages from inducible NO synthase (iNOS)-knockout (KO) mice following αCD40 activation showed reduced tumoristatic activity. CD40 ligation enhanced expression of TNF-α. Macrophage tumoristatic activity following αCD40 treatment was reduced by TNF-α mAb or use of macrophages from TNF-α-KO mice. However, further stimulation of αCD40-activated macrophages with LPS resulted in strong tumoristatic activity that was much less dependent on NO or TNF-α. Taken together, these results suggest that NO and TNF-α are involved in, but not solely responsible for, the antitumour effects of macrophages after activation by CD40 ligation.