Vascular cell adhesion molecule 1 (VCAM-1) is expressed in both endothelial and epithelial cell types, where it contributes to lymphocyte migration to sites of inflammation. Its expression is regulated by cytokines, in part through two κ B-like regulatory elements. Because NF-κ B can be composed of multiple alternative subunits with differential effects on gene expression, the role of different specific NF-κ B family members subunits in VCAM-1 regulation is unknown. In this report, we define the contribution of different NF-κB family members to VCAM-1 gene regulation. We show that both κ B sites in the VCAM-1 enhancer are required to optimally stimulate gene expression, but the enhancer is differentially regulated by specific combinations of NF-κ B subunits. At low concentrations, RelA(p65) acted in concert with the ≈50-kDa product of p105 NF-κ B, NF-κ B1(p50), to stimulate transcription, and at high concentrations, RelA(p65) alone stimulated the VCAM-1 promoter. In contrast, NF-κB2 inhibited functional activation of the VCAM reporter by p65. Consistent with this finding, an additional binding complex was detected by using recombinant NF-κ B2(p49)/RelA(p65) with radiolabeled VCAM κB site probes. Interestingly, the human immunodeficiency virus enhancer responded differently to stimulation by NF-κ B subunits, with optimal response to p49(100)/p65. Analysis of NF-κ B mRNA in human umbilical vein endothelial cells revealed that nfkb1, nfkb2, and relA NF-κ B but not c-rel were induced by tumor necrosis factor α and lipopolysaccharide, which also induce VCAM-1. These data suggest that specific subunits of NF-κ B regulate VCAM-1 and differentially activate other genes in these cells.