Seven days after activation with concanavalin A and irradiated spleen cells, murine CD4⁺ T cells were re-stimulated with lonomycin and phorbol 12-myristate 13-acetate (PMA). IL-2 and IL-4 were determined in the supernatant. When cholera toxin, forskolin together with phosphodlesterase inhibitors or dibutyryl-cAMP were added at the time of re-stimulation, a dose-dependent increase of IL-4 and IL-5 release was noted. IL-2 was down-regulated as reported before. The up-regulatlon of IL-4 and the down-regulation of IL-2 correlated with an increase of IL-4 mRNA and a decrease of IL-2 mRNA as determined by semi-quantitative reverse tratucriptase polymerase chain reaction. Similar results were found with prostaglandin E₂ using PMA and ionomycin or plate-bound anti-CD3 antibody as re-stimulants. These results suggest that, in activated CD4⁺ T cells, cAMP-elevating agents induce a switch of lymphokine production towards a T_h-like phenotype through regulation at the transcriptional level. This is supported by the fact that complex formation between a synthetic nuclear factor of activated T cells (NF-AT) binding site from the IL-2 promoter and nuclear extracts was decreased when cholera toxin was added to re-activated CD4⁺ T cells, suggesting that cholera toxin and cAMP down-regulate IL-2 expression via decreased NF-AT binding. Finally, since IL-4 has been reported to amplify IL-4 release from activated CD4⁺ T cells, the autoinduction of IL-4 may very well function via cAMP.