Dosage-dependent release of ⁴⁵Ca was observed from pre-labeled mouse calvarial bones following treatment with two thiazolidinediones, troglitazone and ciglitazone. Release of ⁴⁵Ca by ciglitazone was decreased by the osteoclast inhibitors acetazolamide, calcitonin (CT), 3-amino-1-hydroxypropylidene-1, 1-bisphosphonate (AHPrBP) and interleukin-4 (IL-4), but not affected by the peroxisome proliferator-activated receptor (PPAR) antagonist, GW 9662, the mitotic inhibitor, hydroxyurea, or indomethacin. Enhanced expression of receptor activator of NFB ligand (RANKL) mRNA and protein and decreased osteoprotegerin (OPG) mRNA and protein were noted following ciglitazone treatment of calvariae. Ciglitazone and RANKL each caused increased mRNA expression of osteoclast markers: calcitonin receptor (CTR), tartrate resistant acid phosphatase (TRAP), cathepsin K, matrix metalloproteinase-9 (MMP-9), integrin 3 and nuclear factor of activated T cells 2 (NFAT2). OPG inhibited mRNA expression of RANKL stimulated by ciglitazone, mRNA expression of osteoclast markers stimulated by ciglitazone and RANKL, and ⁴⁵Ca release stimulated by troglitazone and ciglitazone. Increased expression of IL-1 mRNA by ciglitazone was not linked to resorption stimulated by the thiazolidinedione. Ciglitazone did not increase adipogenic gene expression, but enhanced osteocalcin mRNA in calvariae. In addition to exhibiting sensitivity to OPG, data indicate that stimulation of osteoclast differentiation and activity by thiazolidinediones may occur by a non-PPAR dependent pathway that does not require cell proliferation, prostaglandins or IL-1, but is characterized by an increased RANKL/OPG ratio.