Osteoclasts, cells primarily responsible for bone resorption, differentiate from hematopoietic progenitor cells under the influence of various hormones, cytokines, and differentiation factors. Once fully differentiated, osteoclasts rapidly die in the absence of any survival factor. We have previously shown that tumor necrosis factor alpha (TNF-alpha) promotes the survival of differentiated osteoclasts. The expression of inducible nitric oxide synthase (iNOS) and consequent NO production is often stimulated under inflammatory conditions. In this study, we found that TNF-alpha, but not receptor activator of nuclear factor kappa B ligand and interleukin 1, increased the expression of iNOS both at the mRNA and protein levels. Subsequently, an enhanced NO level was detected both inside the cells and the culture medium of TNF-alpha-stimulated osteoclasts. Blocking NOS activity with L-NAME prevented TNF-alpha-induced NO generation by osteoclasts and the osteoclast survival stimulated by TNF-alpha. The iNOS selective inhibitor L-NIL also suppressed TNF-alpha-induced osteoclast survival, whereas low concentrations of NO releaser NOC-18 were sufficient to promote osteoclast survival. Furthermore, antiapoptotic and caspase suppressive effects of TNF-alpha on osteoclasts were abolished by L-NAME. Our findings indicate that iNOS-dependent NO generation contributes to the survival-promoting function of TNF-alpha in osteoclasts.