AbstractThe nuclear factor kappa B (NF-κB) family of transcription factors orchestrates signal-induced gene expression in diverse cell types. Cellular responses to NF-κB activation are regulated at the level of cell and signal specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate the selective functions of Rel and RelA, two closely related NF-κB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single-cell RNA sequencing revealed marked heterogeneity of Rel- and RelA-specific responses, and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the factors. By rigorously identifying the target genes of each NF-κB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer.