This paper reports on a study of the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, on lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) in RAW 264.7 macrophages and its underlying mechanisms. TSA pretreatment potently diminishes LPS-stimulated nitric oxide (NO) release and both mRNA and protein levels of iNOS in macrophages. The effects of TSA and LPS on transcription factors binding to two LPS-responsive elements within the iNOS promoter, one binding the NF-B site and the other the octamer element, were investigated. Results show that TSA did not alter the LPS-activated NF-B activity demonstrated by the nuclear translocation of p50 and p65, and by a NF-B driven reporter gene expression system. In addition, neither TSA nor LPS changes the expression of Oct-1, a ubiquitously expressed octamer binding protein. However, TSA suppresses the LPS-induced expression of Oct-2, another octamer binding protein, at both mRNA and protein levels. Chromatin immunoprecipitation assays reveal that binding of Oct-2 to the iNOS promoter is enhanced by LPS treatment; however, pretreatment with TSA resulted in loss of this binding. Moreover, forced expression of Oct-2 by transfection of pCG-Oct-2 plasmid restores the TSA-suppressed iNOS expression elevated by LPS stimulation, further indicating that Oct-2 activation is a crucial step for transcriptional activation of the iNOS gene in response to LPS stimulation in macrophages. Taken together, this study demonstrates that TSA diminishes iNOS expression in LPS-treated macrophages by inhibiting Oct-2 expression, and thus reducing the production of NO.