OBJECTIVES: In order to gain new insight into bacterial infection during bone-regenerative treatment using bone morphogenetic proteins (BMPs), we examined the effects of lipopolysaccharide (LPS) on ectopic bone formation induced by BMP-2 and transforming growth factor (TGF)-β1 in mice. METHODS: We implanted collagen sponges containing BMP-2, TGF-β1, and various amounts of LPS into mouse muscle tissues. Lump-like masses in which ectopic bones developed in mice were processed for microcomputed tomography, DNA microarray, reverse-transcription PCR, and histological analyses. RESULTS: LPS treatment caused a dose-dependent reduction in the volume of ectopic bone. The total volume of ectopic bone induced by BMP-2 + TGF-β1 treatment was reduced by more than 75% in the presence of LPS. Histological analysis of the ectopic bone tissues revealed a significant reduction in total bone volume and bone volume/total volume in response to LPS. LPS treatment significantly increased the osteoblast number and osteoid volume, while the osteoclast number did not change. Since LPS induced production of TNF-α and IL-1β in lump-like masses, we implanted collagen sponges containing BMP-2 and TGF-β1 with or without LPS into TNF-α- or IL-1α/β-deficient mice. LPS treatment reduced the volume of ectopic bones in TNF-α-deficient mice but not in IL-1α/β-deficient mice. Furthermore, collagen sponges containing IL-1β reduced ectopic bone formation by BMP-2 and TGF-β1 in wild-type mice to the same extent as LPS treatment did. CONCLUSIONS: LPS suppresses the ectopic bone formation induced by BMP-2 and TGF-β1 through IL-1β production.