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Our poor understanding of how inflammatory mediators can affect osteoblast behavior led us to investigate the tumor necrosis factor (TNF)α-induced focal adhesion kinase (FAK) and Src phosphorylation.MC3T3-E1 pre-osteoblast cells were harvested at specific time points after either TNFαtreatment or RAW267 stimulated conditioned medium, and thereafter cell extracts were prepared for Immunoblotting assay. ELISA detected TNFαcontent at conditioned medium. Tumor necrosis factor-α-neutralizing antibodies also were used.It was possible to show that TNFαprovokes attenuation at Y-phosphorylation of both FAK (at Y397) and Src (at Y416) proteins (P< 0.05), suggesting a decrease in their activities. The very similar profile was observed when osteoblasts were incubated with conditioned medium from lipopolysaccharide (LPS)-stimulated macrophages, it being significantly different than control (FAK and Src,P< 0.05). Nevertheless, in order to validate these findings, we decided to pre-incubate osteoblasts with anti-TNFαneutralizing antibody (2μg ml−1) prior exposing to conditioned medium. Importantly, our results revealed that there was a diminution on those conditioned medium effects when the same biological parameters were evaluated (P< 0.05). Moreover, we also showed that TNFαimpairs osteoblast adhesion, suggesting an interesting role on osteoblast performance.Altogether, these results suggest that LPS-stimulated macrophage mediators attenuate both FAK and Src activations in osteoblast, suggesting a novel role for TNFαon osteoblast performance.