Osteomyelitis is an inflammatory disease of the bone that is characterized by the presence of necrotic bone tissue and increased osteoclast activity. Staphylococcus aureus is responsible for approximately 80% of all cases of human osteomyelitis. While the disease is especially difficult to treat, the pathogenesis of S. aureus-induced osteomyelitis is poorly understood. Elucidating the molecular mechanisms by which S. aureus induces osteomyelitis could lead to a better understanding of the disease and its progression and development of new treatments. Osteoblasts can produce several soluble factors that serve to modulate the activity or formation of osteoclasts. Receptor activator of NF-kappaB ligand (RANK-L) and prostaglandin E(2) (PGE(2)) are two such molecules which can promote osteoclastogenesis and stimulate bone resorption. In addition, previous studies in our laboratory have shown that osteoblasts produce inflammatory cytokines, such as interleukin 6, following infection with S. aureus, which could induce COX-2 and in turn PGE(2), further modulating osteoclast recruitment and differentiation. Therefore, we hypothesized that following infection with S. aureus, osteoblasts will express increased levels of RANK-L and PGE(2). The results presented in this study provide evidence for the first time that RANK-L mRNA and protein and PGE(2) expression are upregulated in S. aureus-infected primary osteoblasts. In addition, through the use of the specific COX-2 inhibitor NS 398, we show that when PGE(2) production is inhibited, RANK-L production is decreased. These data suggest a mechanism whereby osteoblasts regulate the production of RANK-L during infection.