Fibronectin (FN) plays a role in various biological processes such as fibrosis and tumor metastasis. In this study, we investigated the regulation of FN gene expression by NF-kappaB transcription factor. Transient expression of NF-kappaB p65 increased FN promoter activity in rat hepatocytes. Deletion analysis of FN promoter localized the NF-kappaB-responsive region at the position between -1214 and -1126. Mutations in a putative NF-kappaB element (5(')-GAGAATTTCC-3(')) at -1180 blocked most of the p65-induced promoter activity. Electromobility shift assays showed that the expression of p65 induced the binding of the p65/p65 homodimer to the NF-kappaB site at -1180. Stably p65-expressing cells showed increase of promoter activity, FN protein, and its mRNA levels over control cells. Furthermore, treatment of cells with interleukin-1beta, a NF-kappaB-stimulating cytokine, also increased promoter activity, FN production, and mRNA levels. These results show that NF-kappaB activates FN gene expression by binding to the responsive element at -1180 as the p65/p65 homodimer in rat hepatocytes.