The NF-κB family of transcription factors plays a critical role in numerous cellular processes, particularly the immune response. Our understanding of how the different NF-κB subunits act coordinately to regulate gene expression is based on a limited set of genes. We used genome-scale location analysis to identify targets of all five NF-κB proteins before and after stimulation of monocytic cells with bacterial lipopolysaccharide (LPS). In unstimulated cells, p50 and p52 bound to a large number of gene promoters that were also occupied by RNA polymerase II. After LPS stimulation, additional NF-κB subunits bound to these genes and to other genes. Genes that became bound by multiple NF-κB subunits were the most likely to show increases in RNA polymerase II occupancy and gene expression. This study identifies NF-κB target genes, reveals how the different NF-κB proteins coordinate their activity, and provides an initial map of the transcriptional regulatory network that underlies the host response to infection.