Previous studies have implicated CDV in the pathogenesis of Paget's disease; however, there has been no direct evidence that CDV can infect human cells. We studied the effects of CDV on osteoclastogenesis in vitro and showed that CDV had a dose-dependent effect on osteoclastogenesis, through a possible mechanism involving activation of NF-κB and sequestosome 1/p62. Introduction: Paget's disease is characterized by a dramatic increase in size and number of osteoclasts. The etiology of the disorder is still unclear; however, evidence points to either a viral infection or a genetic susceptibility or a combination of both. Previously, we have shown that canine distemper virus (CDV) RNA is present in Pagetic bone. However, the effects of CDV on human osteoclast formation in vitro have not been studied previously. Materials and Methods: Replicate cultures (n = 5) of purified human osteoclast precursors were infected with increasing doses of CDV and cultured on dentine slices for 14 days. Osteoclasts were stained for TRACP, and the dentine slices were examined for evidence of resorption. Control cells were incubated in the absence of virus. In each case, 10 high-power microscopy fields were analyzed. Immunocytochemical analyses were performed for p65, Gab2, sequestosome 1/p62, and ubiquitin. Results: CDV dose-dependently increased osteoclast number and size (p <0.0001, ANOVA), and there was a concomitant increase in resorption (p <0.0001, ANOVA). CDV infection induced nuclear translocation of p65 and led to a dramatic increase in sequestosome 1/p62 and ubiquitin expression. Conclusions: These results provide the first conclusive proof that CDV can infect and replicate in human osteoclast precursors, raising possible zoonotic implications for CDV. The increased osteoclastogenesis is accompanied by NF-κB and sequestosome 1/p62 activation. This study provides further evidence for the possible role of paramyxoviruses in the pathogenesis of Paget's disease. © 2006 American Society for Bone and Mineral Research.