Several clinical studies have shown that bone loss may be attributed to osteoclast recruitment induced by mediators of inflammation. In different experimental paradigms we have recently demonstrated that estrogen exhibits antiinflammatory activity by preventing the induction of inducible nitric oxide synthase (iNOS) and other components of the inflammatory reaction. To verify whether this could explain the estrogen-dependent blockade of osteoporosis, we investigated the effect of ovariectomy in mice in which iNOS activity had been blunted by genetic or pharmacological manipulation. The consequences of iNOS blockade were evaluated initially on bone formation and resorption by histomorphometric analysis. The proximal tibiae of mice with iNOS genotypes revealed that 32 d after ovariectomy bone volume and bone formation rate were significantly decreased, and osteoclast surface was increased. Conversely, in iNOS knockout (iNOSKO) and wild-type (WT) mice treated with a specific inhibitor of iNOS, N-iminoethyl-L-lysine, ovariectomy did not result in bone depletion. In WT mice, ovariectomy also affected bone formation, as shown by a decreased mineral apposition rate. Also in this case, iNOS inactivation prevented the effect of ovariectomy. Immunocytochemical analysis showed that after ovariectomy iNOS protein accumulates in chondrocytes, and a significant increase in nitrotyrosine and poly(ADP-ribose) synthetase staining was observed in the femur metaphyses. The increase in nitrotyrosine and poly(ADP-ribose) synthetase formation induced by ovariectomy was significantly reduced in sections from iNOSKO mice. These data indicate that in WT mice the observed induction of iNOS has functional relevance, because it leads to overproduction of nitric oxide and accumulation of highly reactive molecules, triggering a local inflammatory reaction. These inflammatory foci attract cytokines, well known actors in the mechanism of osteoclastogenesis. In iNOSKO mice the measurements of IL-1 beta, IL-6, and TNFalpha plasma levels showed that ovariectomy fails to elicit the increase observed in WT animals and suggests that iNOS plays a primary role in the protective effects of estrogens. To further support this hypothesis, we show that estradiol-dependent activation of estrogen receptor-alpha blocks phorbol 12-acetate 13-myristate-induced transcription of iNOS promoter in transfected cells, thus demonstrating that the promoter of iNOS is under estrogen negative control. Our findings point to a key role of iNOS in mediating the negative effects of estrogen depletion on bones and provide a novel mechanistic explanation for the effects of menopause in osteoporosis and possibly also in other diseases in which the inflammatory component is elevated.