Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor family that is implicated in apoptosis, proliferation, migration, and inflammation. We describe our findings showing that TWEAK mediated the differentiation of RAW264.7(RAW) monocyte/macrophage cells into multinuclear, functional osteoclasts. The effect of TWEAK was direct and not mediated by the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) as shown by the use of TWEAK- or RANKL-neutralizing antibodies and by osteoprotegerin, a decoy receptor for RANKL. Recently, fibroblast growth factor-inducible 14 (Fn14) was suggested to be a receptor for TWEAK. We show that the Fn14/TWEAK receptor (TweakR) was not responsible for the osteoclastic effect of TWEAK on RAW cells. Flow cytometry analysis did not reveal the expression of Fn14/TweakR on RAW cells. Moreover, Fn14/TweakR-neutralizing antibodies did not block TWEAK- induced RAW cell differentiation into osteoclasts. This indicated that a second TweakR, TweakR2, exists on RAW cells and is responsible for mediating TWEAK- induced differentiation. We next compared the signaling pathways that are activated by the two receptors. TWEAK binding to TweakR2 activated the NF-kappaB, mitogen-activated protein kinase and c-Jun N-terminal kinase signaling cascades in RAW cells. In contrast, TWEAK binding to Fn14/TweakR activated the NF-kappaB and c-Jun N-terminal kinase pathways but induced only a weak activation of MAPK in HT-29 human colon adenocarcinoma cells expressing endogenous Fn14/TweakR. We propose that the biological effects of TWEAK are mediated by binding to one of at least two distinct receptors that induce differential activation of downstream signaling pathways.