The role of inducible nitric oxide synthase (iNOS) in bone development and bacterially induced periodontal bone loss was examined using mice with targeted mutation of the iNOS gene. Femurs of iNOS KO mice showed 30% and 9% higher bone mineral density compared to wild type (WT) at 4 and 9 weeks of age, respectively. Micro-computed tomography revealed that cortical thickness and cortical bone density is increased in the absence of iNOS, while trabecular bone thickness and bone density remains unchanged. Histochemical analysis using TRAP staining showed that osteoclast numbers are lower by 25% in iNOS KO femurs compared to WT femurs. When bone marrow cells were stimulated with M-CSF and RANKL in vitro, iNOS KO cultures developed 51% fewer TRAP-positive multinuclear cells compared to WT cultures. When similar cultures were grown on dentine discs, resorption pit area was decreased by 54% in iNOS KO cultures. Gene expression studies showed that iNOS expression is induced by M-CSF and RANKL in WT bone marrow cultures, while no iNOS transcript was detected in iNOS KO. No compensatory change was detected in the expression of neuronal or endothelial NOS isoforms. There was no difference in RANK and osteoprotegerin expression between iNOS KO and WT bone marrow cultures after M-CSF and RANKL-treatment, while Traf6 expression was significantly lower in the absence of iNOS. In the alveolar bone of the maxilla, the distance between the cementoenamel junction and the alveolar bone crest was larger in iNOS KO compared to WT mice from 6 to 14 weeks of age, indicating a developmental effect of iNOS in oral tissues. Oral administration of the periodontal pathogen Porphyromonas gingivalis caused alveolar bone loss in the maxilla of WT mice, but failed to do so in iNOS KO mice. Expression of the osteoclast marker cathepsin K was 25% lower in iNOS KO alveolar bone. These data indicate that iNOS promotes bone resorption during bone development as well as after bacterial infection, and that iNOS is an important signal for normal osteoclast differentiation.