We have delineated a positive regulatory element in the interleukin-2 receptor alpha-chain gene (IL-2R alpha) between positions -299 and -243 that can potently activate a heterologous (herpesvirus thymidine kinase [tk]) promoter in phorbol myristate acetate (PMA)-induced Jurkat T cells and is functional when cloned in either orientation. This enhancerlike element contains a site (-268/-257) that can bind NF-kappa B; however, unlike the immunoglobulin kappa gene kappa B enhancer element, the IL-2R alpha kappa B-like site alone can only weakly activate a heterologous promoter. Adjacent 5' and 3' sequences also weakly activate the tk-CAT vector, but constructs combining the IL-2R alpha kappa B-like site plus adjacent 5' and 3' sequences potently activate gene expression. This combination of regions is essential for potent PMA-induced transcription from the tk promoter. Experiments using constructs in which IL-2R alpha upstream sequences are sequentially deleted suggested that there is a region 5' of position -299 which can suppress IL-2R alpha promoter and/or enhancer activity. Thus, it is possible that both positive and negative elements may be important in the regulation of IL-2R alpha gene transcription.