To understand the basis for T-cell-specific induction of interleukin 2 (IL-2), we have analyzed nuclear factors from the Jurkat T-lymphoid leukemia cell, which can be induced to secrete IL-2. We have used an electrophoretic mobility shift assay to examine binding of proteins to the upstream regulatory region, before and after activation with mitogens. We find two types of binding sites. One resembles an inducible enhancer element, but the protein that recognizes it is found in non-T cells and is unlikely to determine T-cell-specific expression of IL-2. A second site negatively regulates expression in resting T cells. A complex that binds to a DNA fragment containing this site is modified only when IL-2 is expressed, and it lies near a specific inducible DNase hypersensitive region. We suggest that negative regulation at this site, mediated by its associated protein(s), may contribute to the cell-specific expression of IL-2.