Receptor activator of nuclear factor-kappaB ligand (RANKL) is a key factor necessary for osteoclast differentiation and activation. Mutations within the TNF-like core domain of RANKL have been recently reported in patients with osteoclast-poor autosomal recessive osteopetrosis. However, the functional consequence owing to RANKL mutations has not been well characterized. Here we describe the functional propensity of RANKL mutants in osteoclast differentiation and their impact on RANKL-mediated signaling cascades. Recombinant RANKL (rRANKL) mutants within the TNF-like core domain exhibited diminished osteoclastogenic potential as compared with wild-type rRANKL1 encoding the full TNF-like core domain [amino acids (aa) 160-318]. Consistent with the insufficient activities on osteoclastogenesis, rRANKL mutants showed reduced activation of nuclear factor-kappaB, IkappaBalpha degradation, and ERK phosphorylation. In addition, we found that rRANKL mutants interfered with wild-type rRANKL-induced osteoclastogenesis with deletion mutant rRANKL5 (aa 246-318) exhibiting the greatest inhibitory effect. The same mutant also significantly reduced wild-type rRANKL1 (aa 160-318)-induced osteoclastic bone resorption in vitro. BIAcore assays demonstrated that rRANKL5 alone, lacking the AA'' and CD loops, weakly binds to receptor activator of nuclear factor-kappaB (RANK). Intriguingly, preincubation of mutant rRANKL5 with rRANKL1 before exposure to RANK enhanced the maximal binding level to RANK, indicating that rRANKL5 forms hybrid trimeric complexes with rRANKL1. Furthermore, RANKL mutant mimicking human RANKL V277 mutation in patients, impairs osteoclast differentiation and signaling. Taken together, these data lend support to the notion that the TNF-like core domain of RANKL contains structural determinants that are crucial for osteoclast differentiation and activation, thus providing a possible mechanistic explanation for the observed phenotype in osteopetrotic patients harboring RANKL mutations.