We analyzed tumor necrosis factor (TNF) responses of human umbilical artery and vein endothelial cells (HUAECs and HUVECs) in organ and cell culture. In organ culture, TNF induced expression of E-selectin, VCAM-1, and ICAM-1 on HUVECs but only ICAM-1 on HUAECs. Activation of nuclear factor-kappaB, c-jun, and ATF2 by TNF was comparable in HUAECs and HUVECs, whereas binding of transcription factors and p300 co-activator to the E-selectin enhancer was lower in HUAECs compared to HUVECs. In cell culture, HUAECs rapidly acquired inducible E-selectin and VCAM-1 whereas ICAM-1 inducibility decreased. Culture of HUVECs rapidly decreased TNF responses of all three genes. By 72 hours in cell culture, TNF-treated HUVECs and HUAECs showed comparable adhesion molecule induction and transcription factor binding to the E-selectin enhancer. Freshly isolated HUAECs expressed higher levels of Kruppel-like factor 2 (KLF2) than HUVECs, consistent with greater KLF2 induction by arterial levels of shear stress in vitro. KLF2 expression decreased rapidly in both cell types during culture. Transduction of HUVECs with KLF2 reduced TNF-mediated induction of E-selectin and VCAM-1 while increasing ICAM-1 induction and reduced transcription factor/co-activator binding to the E-selectin enhancer. In conclusion, the differential responses of HUAECs and HUVECs to TNF in organ culture correlate with transcription factor/co-activator binding to DNA and converge during cell culture. Flow-induced expression of KLF2 contributes to the in situ responses of HUAECs but not of HUVECs.