Cytokine-induced expression of the E-selectin gene requires the promoter binding and interaction of the transcription factors NF-kappa B and ATF. Here we have further analyzed the E-selectin promoter and revealed an additional region (nucleotides -140 to -105 [-140/-105]) which is essential in controlling promoter activation by cytokines. We identified high-mobility-group protein I(Y) [HMG-I(Y)] interacting specifically at two sites within this region. We noted that one of the HMG-I(Y)-binding sites overlaps a sequence element (-127/-118) diverging at only one position from the NF-kappa B consensus binding sequence. This led us to ask whether the -127/-118 element represents a second functional NF-kappa B-binding site within the E-selectin promoter. Using specific antisera, we show that p50, p65, and, interestingly, RelB are components of the complex interacting at this site. Mutational analysis of the -127/-118 NF-kappa B site indicates that both NF-kappa B and HMG-I(Y) binding at this site are essential for interleukin-1 induction of the promoter. We demonstrate that the binding affinity of the p50 subunit of NF-kappa B to both NF-kappa B sites within the E-selectin promoter is significantly enhanced by HMG-I(Y). In addition, an essential role for cooperative interaction between the two NF-kappa B complexes is shown by the requirement for both NF-kappa B sites to mediate E-selectin promoter activation by interleukin-1 and p50/p65 expression. We conclude that HMG-I(Y) mediates binding of a distinct NF-kappa B complex at two sites within the E-selectin promoter. Furthermore, a unique cooperativity between these NF-kappa B complexes is essential for induced E-selectin expression. These results suggest mechanisms by which NF-kappa B complexes are involved in specific gene activation.