Abstract The alternative NF-κB pathway consists predominantly of NF-κB-inducing kinase (NIK), IκB kinase α (IKKα), p100/p52, and RelB. The hallmark of the alternative NF-κB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF-κB activation in bone biology, however, is not well understood. To elucidate the role of the alternative pathway in bone homeostasis, we first analyzed alymphoplasic (aly/aly) mice, which have a defective NIK and are unable to process p100, resulting in the absence of p52. We observed increased bone mineral density (BMD) and bone volume, indicating an osteopetrotic phenotype. These mice also have a significant defect in RANKL-induced osteoclastogenesis in vitro and in vivo. NF-κB DNA-binding assays revealed reduced activity of RelA, RelB, and p50 and no binding activity of p52 in aly/aly osteoclast nuclear extracts after RANKL stimulation. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100−/− mice, which specifically lack the p100 inhibitor but still express p52. p100−/− mice have an osteopenic phenotype owing to the increased osteoclast and decreased osteoblast numbers that was rescued by the deletion of one allele of the relB gene. Deletion of both allele of relB resulted in a significantly increased bone mass owing to decreased osteoclast activity and increased osteoblast numbers compared with wild-type (WT) controls, revealing a hitherto unknown role for RelB in bone formation. Our data suggest a pivotal role of the alternative NF-κB pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis and the importance of RelB in both bone formation and resorption. © 2010 American Society for Bone and Mineral Research