IL-2 gene transcription occurs in an activation-dependent manner in T cells responding to TCR and CD28 activation. One of the critical events leading to increased IL-2 transcription is an alteration in chromatin structure across the 300-bp promoter region of the gene. We initially showed that IL-2 gene transcription in CD4+ primary T cells is dependent on the NF-κB family member, c-Rel, but not RelA. We found that c-Rel is essential for global changes in chromatin structure across the 300-bp IL-2 promoter in response to CD3/CD28 in primary CD4+ T cells, but not in response to pharmacological signals, paralleling the requirement for c-Rel in IL-2 mRNA and protein accumulation. Interestingly, measurement of activation-induced localized accessibility changes using restriction enzyme digestion revealed that accessibility close to the c-Rel binding site in the CD28RR region of the promoter is specifically dependent on c-Rel. In contrast, restriction enzyme sites located at a distance from the CD28RR behave independently of c-Rel. These results suggest a nonredundant role for c-Rel in generating a correctly remodeled chromatin state across the IL-2 promoter and imply that the strength of the signal determines the requirement for c-Rel.