Recent evidence suggests that the production of nitric oxide (NO) may have important roles in the regulation of osteoblast and osteoclast metabolism. The present study was performed to investigate the effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) on the expression of inducible NO-synthase (iNOS) and to measure high-output production of NO by primary rat osteoblasts and osteoblastic cell lines ROS 17/2.8, MC3T3-E1 and MG-63. In addition, we have investigated if NO may mediate some of the effects of these cytokines on osteoblast metabolism. Northern blots and immunocytochemistry revealed time-dependent iNOS messenger RNA and protein expression in primary rat osteoblasts in response to cytokine treatment. Reverse transcription polymerase chain reaction amplified an 807-base pair (bp) product from ROS 17/2.8 cells, which had a size and restriction enzyme-cut pattern identical to that predicted for authentic rat iNOS. Nitrite accumulation in culture medium was induced by IFN-gamma in a time- and dose-dependent manner and inhibited by cotreatment with inhibitors of NOS activity and by dexamethasone. IL-1 beta, TNF-alpha, and bacterial lipopolysaccharide were found to have weak stimulatory effects on nitrite production on their own. However, IL-1 beta and TNF-alpha showed strong synergy with IFN-gamma, but, surprisingly, lipopolysaccharide was found to exert potent inhibitory effects on IFN-gamma-induced nitrite synthesis. Basal production of nitrite and induction of its synthesis was similarly observed with primary rat osteoblasts as well as ROS 17/2.8, MC3T3-E1, and MG-63 cell lines. Cytokine-induced NO production significantly reduced osteoblast activity, as was evidenced by inhibition of DNA synthesis, cell proliferation, alkaline phosphatase activity, and osteocalcin production. The results provide evidence for a basal expression of iNOS activity and show that the iNOS messenger RNA, protein, and enzyme activity are all induced by cytokines across the species. The data further suggest that osteoblast-derived NO may have an important role in mediation of localized bone destruction associated with inflammatory bone diseases such as rheumatoid arthritis.