Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A(1)R, A(2A)R, A(2B)R, and A(3)R). Because A(1)R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A(1)R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A(1)R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the presence of colony stimulating factor-1 and receptor activator of NF-kappaB ligand in the presence or absence of the A(1)R antagonist 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Osteoclast morphology was analyzed in tartrate-resistant acid phosphatase or F-actin-stained samples, and bone resorption was evaluated by toluidine blue staining of dentin. BMCs from A(1)R-knockout mice form fewer osteoclasts than BMCs from WT mice, and the A(1)R antagonist DPCPX inhibits osteoclast formation (IC(50)=1 nM), with altered morphology and reduced ability to resorb bone. A(1)R blockade increased ubiquitination and degradation of TRAF6 in RAW264.7 cells induced to differentiate into osteoclasts. These studies suggest a critical role for adenosine in bone homeostasis via interaction with adenosine A(1)R and further suggest that A(1)R may be a novel pharmacologic target to prevent the bone loss associated with inflammatory diseases and menopause.