We have recently demonstrated that a transcription factor, nuclear factor kappa B (NF-kappa B), plays a key role in the production of cytokine and cell adhesion molecules in osteoblastic cells. In the present study, we investigated the effects of vector-averaged gravity (clinostat rotation) on tumor necrosis factor (TNF)-alpha-induced activation of NF-kappa B in human osteoblastic HOS-TE85 cells. After a 72-hr clinostat culture, the cells were treated with TNF-alpha for 30 min. Electrophoretic mobility shift assay using the nuclear extracts revealed that the DNA-binding activity of NF-kappa B was substantially reduced in clinostat-cultured cells compared with the stationary control. Concomitantly, the transactivation of NF-kappa B was examined by a transfection study. The cells were transfected with a plasmid expressing a luciferase reporter gene driven by multimerized NF-kappa B sites. They were cultured in the clinostat for 48-hr, followed by a 4-hr treatment with TNF-alpha. Clinostat culture resulted in a significant decrease in the luciferase activities, being consistent with the decreased binding of NF-kappa B. These results indicate that exposure of HOS-TE85 cells to vector-averaged gravity impairs NF-kappa B activation by TNF-alpha.