HOXA9 is a homeobox transcription factor expressed in endothelial cells (EC) and its expression is rapidly downregulated during EC activation by inflammatory signals like tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide (LPS). Recently, we have shown that HOXA9 overexpression prevents EC activation by inhibiting NF-kappaB activity, which suggests that HOXA9 downregulation is an essential event for EC activation. The present study is directed towards understanding the mechanism of HOXA9 regulation during EC activation. Here we show that nuclear factor-kappaB (NF-kappaB) activation is an essential step for HOXA9 downregulation. Deletion analyses of HOXA9 promoter in EC and NF-kappaB knockout cells have shown that NF-kappaB is a major transcription factor that is absolutely required for HOXA9 downregulation. Our 5' deletion analysis of HOXA9 promoter shows that NF-kappaB response element is localized within first 400 nucleotides, while minimal basal promoter is within 100 nucleotides upstream of its transcriptional start site. We demonstrate that HOXA9 regulates its own expression by positive feedback mechanism. To define mechanism by which HOXA9 autoregulates its expression, we show that HOXA9 DNA binding and transactivation domains are essential.