Reveromycin A (RM-A), a small natural product isolated from Streptomyces bacteria, is a potential osteoporosis therapeutic in that it specifically induces apoptosis in osteoclasts but not osteoblasts. The purpose of the study presented here was to further elucidate the intracellular mechanisms of RM-A death effects in mature osteoclasts. A specific clone of RAW264.7 murine macrophages that was previously characterized for its ability to acquire an osteoclast nature on differentiation was differentiated in the presence of receptor activator of nuclear factor kappa B ligand (RANKL). Subsequent staining was performed for tartrate-resistant acid phosphatase to confirm their osteoclast character. These osteoclasts were treated with ten micromolar RM-A for 2, 4, 6, 24, and 48 h at a pH of 5.5. Peak apoptosis induction occurred at 4-6 h as measured by caspase 3 activity. Lactate dehydrogenase release assay revealed no significant RM-A-induced necrosis. Western blot analysis of cytoplasmic extracts demonstrated activation of caspase 9 (2.3-fold at 2 h and 2.6-fold at 4 h, each P < 0.05) and no significant changes in Bcl-XL . In nuclear extracts, NFκB levels significantly increased on differentiation with RANKL but then remained constant through RM-A treatment. Over the extended time course studied, RM-A-induced apoptosis in osteoclasts was not accompanied by necrosis, suggesting that RM-A would likely have limited effects on immediate, neighboring bone cell types. This specific cell death profile is promising for potential clinical investigations of RM-A as a bone antiresorptive.