The activation of cytokine genes in response to antigenic stimulation of T cells is mediated by NF-AT proteins. Previous studies have identified two NF-AT proteins, NF-ATp and NF-ATc, that are homologous within a 290 aa domain distantly related to the Rel domain. We have isolated two additional members of this gene family, NF-AT3 and NF-AT4, which encode proteins 65% identical to the other NF-AT proteins within the Rel domain. The four NF-AT genes are transcribed in different sets of tissues that included many sites of expression outside the immune system. The Rel homology domain is sufficient for DNA recognition and cooperative binding interactions with AP-1. Although other members of the Rel family bind DNA as dimers, NF-AT proteins are monomers in solution or bound to DNA. Transfection assays indicate that each of the four NF-AT proteins can activate the IL-2 promoter in T cells.