A decrease of endothelial nitric oxide synthase (NOS-3)-derived NO is the major cause of endothelial dysfunction. In vivo, NOS-3 expression is driven by fluid shear stress (FSS). Besides basal factors such as SP-1 and GATA-isoforms, the krüppel-like transcription factor KLF-2 has been shown to be involved in NOS-3 expression. However, the complex regulation of the gene still is not fully understood. We here have analyzed FSS-dependent nos-3 gene expression in human primary cultured endothelial cells using a plate and cone-viscometer (30 dyne/cm²). Real time RT-PCR, Western blot and IFA were used to assay mRNA and protein levels as well as protein localization. To screen for proteins specifically binding to FSS-sensitive parts of the nos-3 promoter, a combination of affinity purification (a 200 bp promoter-fragment coupled to magnetic beads) followed by mass spectrometry was used. In our experimental model, KLF-2 did not affect fluid shear stress-induced NOS-3 expression. Moreover, the factor did not bind to the fluid shear stress-sensitive part of the nos-3 gene promoter. Mass spectrometric analysis identified the ATP-dependent DNA helicase-2 (subunits 1 and 2) as well as gamma-actin and beta-actin-like protein-2 to specifically bind to the promoter in a FSS-dependent way. The DNA-helicase-2/actin complex may regulate the nos-3 gene rather by facilitating the access of basal transcription factors such as SP-1 than by directly acting as transcriptional activators. This mechanism could define a new regulatory principle at the single gene level.