Effects of vector-averaged gravity on tumor necrosis factor (TNF)-alpha -dependent activation of nuclear factor kappa B (NF-kappaB) in human osteoblastic HOS-TE85 cells were investigated by culturing the cells using clinostat rotation (clinorotation). Cell cultures were rotated for 72 h at 40 rpm in a clinostat. At the end of clinorotation, the cells mere treated with TNF-alpha for 30 min under stationary conditions. Electrophoretic mobility shift assays revealed that TNF-alpha -dependent activation of NF-kappaB was markedly reduced in the clinorotated cells when compared with the cells in control stationary cultures or after horizontal rotation (motional controls). The NF-kappaB-dependent transactivation was also impaired in the clinorotated cells, as evidenced by a transient transfection assay with a reporter plasmid containing multimerized NF-kappaB sites. Consistent with these findings, the TNF-alpha -dependent induction of endogenous NF-kappaB-responsive genes p105, I kappaB-alpha, and IL-8, was significantly attenuated in clinorotated cells. These results demonstrate that vector-averaged gravity inhibits the responsiveness of osteoblasts to TNF-alpha by repressing NF-kappaB activation.