Chemically modified tetracyclines (CMTs) are thought to inhibit bone resorption primarily through their ability to inhibit matrix metalloproteinases (MMPs). We have previously demonstrated that some tetracycline compounds (TCs) induce apoptosis in mature rabbit osteoclasts and inhibit osteoclastic resorption in mouse osteoblast/marrow co-cultures in vitro. In this report, we now show that non-antibiotic analogues of doxycycline (CMT-3) and minocycline (CMT-8) are potent inhibitors of osteoclastogenesis in vitro from human peripheral blood mononuclear cells (PBMC) stimulated with macrophage colony stimulating factor (MCSF) and receptor activator of NF-kappaB ligand (RANKL), through an action that is independent of osteoblast-osteoclast interactions. Osteoclast formation over 20 days was completely abrogated when CMT-3 or CMT-8 were included in PBMC cultures at a concentration of 250 ng/ml, although doxycycline at this concentration reduced osteoclast formation to ca. 50% of control. CMT-3 and CMT-8 also significantly induced apoptosis over 24 h in mature osteoclasts generated over 20 days when added to cultures at 5 microg/ml or more. In a time-course experiment, apoptosis was evident after a delay of 1-2 h following treatment of mature osteoclasts with CMT-3 at 20 microg/ml. The broad-spectrum MMP inhibitor BB94 (Batimastat) did not recapitulate the apoptosis induced by CMT-3, even at a concentration where MMP-13 activity was completely inhibited. There was no evidence for an anabolic effect of any of the TCs on osteoblast lineage cells in a calcifying fibroblastic colony (CFU-f) formation assay, where CMT-3 partially inhibited CFU-f formation at 5 microg/ml. Our data indicate that inhibition of osteoclast formation and induction of osteoclast apoptosis are pharmacologically significant actions of CMTs in inhibiting bone resorption, and that osteoclast apoptosis cannot be attributed to the ability of CMTs to inhibit MMPs or to actions mediated by osteoblastic lineage cells.