To characterize the NF-kappa B binding factor in molecular terms and to facilitate the cloning of its gene, we have purified this protein from bovine spleen tissue. We have found it is a 42,000-dalton protein that exists in solution as a dimer. We were able to use the purified protein to show that the same polypeptide is able to recognize sites important for activation of genes in either B- or T-lymphocytes. Moreover, we were able to define a consensus sequence which allows ascertainment of a wider variety of sequences that are capable of interacting with this protein. The implication of the same protein in gene regulation in two different lineages of lymphoid cells reveals an unexpected unity in the mechanism of gene expression during B- and T-lymphocyte activation. This also suggests that other regulatory events must participate with NF-kappa B activation in determining B- or T-cell-specific expression.