Proliferating human medullary thymocytes can exhibit characteristic T helper cell type 1 cytokine responses exemplified by the immediate early expression of interleukin-2, interferon-gamma, tumor necrosis factor-alpha, and lymphotoxin-beta. Here we report that cAMP-mediated attenuation of the transcription of T helper-1-specific cytokine genes in human medullary thymocytes correlates with the induction of the cAMP-mediated transcriptional repressor ICER (inducible cAMP early repressor). We show that ICER binds specifically to several NFAT/AP-1 (nuclear factor of activated T cells/activating protein-1) composite DNA sites essential for the activation of the interleukin (IL)-2 promoter as well as to a homologous DNA motif present in the proximal segment of the interferon-gamma promoter. In the presence of the minimal NFAT DNA-binding domain, which is sufficient for both DNA binding and AP-1 complex formation, ICER and NFAT form NFAT/ICER ternary complexes on several NFAT/AP-1 DNA composite sites previously identified as essential for the expression of the immunoregulatory cytokines such as IL-2, IL-4, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha. In extracts prepared from human medullary thymocytes treated with forskolin and ionomycin, these composite sites bind endogenously expressed ICER either singly or in complexes. Moreover, in Jurkat cells, ectopically expressed ICER represses transcription from NFAT-mediated, phorbol ester/ionophore-activated IL-2, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha promoters. We present evidence that ICER interactions with NFAT/AP-1 composite DNA sites correlate with its ability to repress transcription. These findings provide further insight into the mechanisms involved in cAMP-mediated transcriptional attenuation of cytokine expression.