Receptor activator of nuclear factor-κB ligand [RANK ligand (RANK-L)] stimulates mature osteoclasts to resorb bone, a process associated with NF-κB activation. RANK-L also prompts macrophages to develop the osteoclast phenotype. Although NF-κB is essential for osteoclast differentiation, it is not known whether RANK-L activates this transcription complex in osteoclast precursors. We report that RANK-L rapidly induces NF-κB activation in both authentic osteoclast precursors, namely bone marrow macrophages, and RAW 264.7 cells, a murine macrophage line also capable of RANK-L-mediated osteoclastogenesis. Supershift studies reveal the RANK-L-induced DNA binding moiety contains p50/p65, the most common NF-κB complex. Subcellular translocation of p50 and p65 subunits is confirmed by Western blots and immunofluorescence analysis. RANK-L activates NF-κB in both bone marrow macrophages and RAW 264.7 cells by serine phosphorylation of IκBα within 5 min, resulting in rapid IκBα degradation and resynthesis. Attesting to function, RANK-L treatment of RAW 264.7 cells transiently transfected with a plasmid containing NF-κB consensus elements linked to luciferase greatly enhances reporter activity. Our data suggest that activation of the NF-κB pathway is an integral component of RANK-L-induced osteoclast differentiation.