IL-2 gene expression is regulated by the cooperative binding of discrete transcription factors to the IL-2 promoter/enhancer and is predominantly controlled at the transcriptional level. In this study, we show that in normal T cells, the− 180 site (− 164/− 189) of the IL-2 promoter/enhancer is a p-cAMP-responsive element-binding protein (p-CREB) binding site. Following activation of the T cells through various membrane-initiated and membrane-independent pathways, protein kinase C (PKC)-θ phosphorylates CREB, which subsequently binds to the− 180 site and associates with the transcriptional coactivator p300. Rottlerin, a specific PKC-θ inhibitor, diminished p-CREB protein levels when normal T cells were treated with it. Rottlerin also prevented the formation of p-CREB/p300 complexes and the DNA-CREB protein binding. Cotransfection of fresh normal T cells with luciferase reporter construct driven by two tandem− 180 sites and a PKC-θ construct caused a significant increase in the transcription of the reporter gene, indicating that this site is functional and regulated by PKC-θ. Cotransfection of T cells with a luciferase construct driven by the− 575/+ 57 region of the IL-2 promoter/enhancer and a PKC-θ construct caused a similar increase in the reporter gene transcription, which was significantly limited when two bases within the− 180 site were mutated. These findings show that CREB plays a major role in the transcriptional regulation of IL-2 and that a major pathway for the activation of CREB and its subsequent binding to the IL-2 promoter/enhancer in normal T cells is mediated by PKC-θ.