Interleukin-3 (IL-3) is involved in proliferation and differentiation of hematopoietic progenitor cells. Its expression is subject to precise, tissue-specific regulation, and has been studied extensively in the gibbon T-cell line MLA 144 by a combination of functional assays and DNA binding experiments. To extend these studies, the gibbon IL-3 promoter was cloned and in vivo footprinting of the gibbon and human IL- 3 proximal promoters was performed. The gibbon IL-3 promoter was found to be highly homologous to its human counterpart and both promoters yielded identical in vivo footprints after induction of IL-3 synthesis. In particular, we observed specific protection of three guanines over a core sequence TGTGGTTT (IF-1IL3) that had not been recognized in previous studies. IF-1IL3 is not found in other cytokine promoters, but it is conserved in the IL-3 promoter of several species and is similar to a recurring motif in viral and T-cell-specific cellular enhancers. IF-1IL3 binds a specific complex in MLA 144 and Jurkat nuclear extracts in vitro, which shares the same specificity as the complex bound by the polyoma virus and T-cell receptor delta enhancers. Mutation of the three guanines in IF-1IL3 core sequence disrupts binding in vitro and abrogates the ability of the IL-3 promoter to mediate inducible expression in T cells. Although IF-1IL3 is necessary for IL-3 expression, it is not sufficient: a truncated IL-3 promoter with an intact IF-1IL3 site but no other activator sites is transcriptionally silent. These studies describe a new regulatory element within the IL-3 promoter that is essential for expression and conserved between species.