The human tumor necrosis factor alpha (TNF-α) gene is one of the earliest genes expressed upon the activation of a T or B cell through its antigen receptor. Previous experiments have demonstrated that in stimulated T cells, a TNF-α promoter element, κ3, which binds NFATp, is required for the cyclosporin A-sensitive transcriptional activation of the gene. Here, we demonstrate that a cyclic AMP response element (CRE), which lies immediately upstream of the κ3 site, is also required for induction of TNF-α gene transcription in T cells stimulated by calcium ionophore or T-cell receptor ligands. The CRE binds ATF-2 and Jun proteins in association with NFATp bound to κ3. These proteins bind noncooperatively in vitro; however, the transcriptional activity of the CRE/κ3 composite site is dramatically higher than the activity of the κ3 site alone, indicating that the two sites cooperate in vivo. This study is the first demonstration of a role for ATF-2 in TNF-α gene transcription and of a functional interaction between ATF-2/Jun and NFATp. This novel pairing of NFATp with ATF-2/Jun may account for the specific and immediate pattern of TNF-α gene transcription in stimulated T cells.