Tumor necrosis factor (TNF) is a pleiotropic cytokine known to regulate cell growth, viral replication, inflammation, immune system functioning, angiogenesis, and tumorigenesis. These effects are mediated through two different receptors, TNFR1 and TNFR2 (also called p60 and p80, respectively), with p60 receptor being expressed on all cell types and p80 receptor only on cells of the immune system and on endothelial cells. Although the role of p60 receptor in TNF signaling is well established, the role of p80 is less clear. In this report, by using macrophages derived from wild-type mice (having both receptors) and mice in which the gene for either p60 (p60(-/-)), or p80 (p80(-/-)), or both (p60(-/-) p80(-/-)) receptor have been deleted, we have redefined the role of these receptors in TNF-induced activation of nuclear factor (NF)-kappa B and of mitogen-activated protein kinases. TNF activated NF-kappa B in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. These results correlated with the I kappa B alpha degradation needed for NF-kappa B activation. We also found that TNF activated c-Jun N-terminal protein kinase in a dose- and time-dependent manner in wild-type macrophages but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF activated p38 MAPK and p44/p42 MAPK in wild-type but not in p60(-/-), p80(-/-), or p60(-/-) p80(-/-) macrophages. TNF induced the proliferation of wild-type macrophages, but for p60(-/-) and p80(-/-) macrophages proliferation was lower, and in p60(-/-) p80(-/-) it was absent. Overall, our studies suggest that both types of TNF receptors are needed in macrophages for optimum TNF cell signaling.