Tissue inhibitor metalloproteinases 1 (TIMP-1) and 2 have been reported to inhibit bone resorption. However, here, we report the direct action of both TIMP-1 and TIMP-2 on isolated rabbit mature osteoclasts to stimulate their bone-resorbing activity at significantly lower concentrations (approximately ng/ml) than those (approximately microg/ml) required for the inhibition of bone resorption. The cell population used in this study consisted of a mature osteoclast population with >95% purity. TIMP-1 (approximately 50 ng/ml) and TIMP-2 (approximately 8-10 ng/ml) increased the pit area excavated by the isolated mature osteoclasts. The stimulatory effects of TIMPs were abolished by simultaneous addition of anti-TIMP antibodies. At higher concentrations, the stimulation of bone resorption decreased reversely to the control level. The magnitude of the stimulatory effect of TIMP-2 was more than that of TIMP-1. Metalloproteinase inhibitors such as BE16627B and R94138 could not replace TIMPs with respect to the bone-resorbing activity, suggesting that the osteoclast-stimulating activity of TIMPs was independent of the inhibitory activity on matrix metalloproteinases (MMPs). TIMPs stimulated tyrosine phosphorylation of cellular proteins in the isolated mature osteoclasts. Both herbimycin A, an inhibitor of tyrosine kinases, and PD98059 and U0126, inhibitors of mitogen-activated protein kinase (MAPK), completely blocked the TIMP-induced stimulation of osteoclastic bone-resorbing activity. On the plasma membrane of osteoclasts, some TIMP-2-binding proteins were detected by a cross-linking experiment. These findings show that TIMPs directly stimulate the bone-resorbing activity of isolated mature osteoclasts at their physiological concentrations and that the stimulatory action of TIMPs is likely to be independent of their activities as inhibitors of MMPs.