Abstract IL-4 is a pleiotropic cytokine whose expression is limited to a subset of activated T cells and cells of the basophil/mast cell lineage. It plays a key role in regulating many immune responses; however, little is known about the intracellular signaling events that lead to the selective and transient IL-4 expression in either of these cell types. In this study, the molecular basis of stimulation-dependent transcription in T cells was explored. To identify cis elements that regulate IL-4 gene transcription, various amounts of the 5' flanking region of the murine IL-4 gene were linked to a chloramphenicol acetyl transferase (CAT) reporter gene and tested for the ability to modulate CAT gene transcription in PMA-stimulated EL-4 T cells. These experiments indicate that multiple positive and negative-acting elements contribute to the overall level of IL-4 transcription. These elements are located both proximal and distal to the transcription initiation site (TIS). An activation responsive element is located within 87 bp of the IL-4 gene TIS. This sequence is sufficient to confer responsiveness to PMA-mediated signals and results in a 10- to 20-fold induction of CAT reporter gene activity compared to activity detected in unstimulated cells. Proteins that specifically bind sequences within this region (-88 to -60) are detected in both unstimulated and stimulated EL-4 T cell nuclear extracts. An additional DNA-protein interaction is detected only when extracts from stimulated cells are analyzed. Base substitutions within the -88 to -60 sequence affect both transactivation function and protein/DNA interactions and demonstrate that sequences between -78 and -69 bp are critical. Together, these data support a model in which T cell activation signals stimulate binding of a nuclear protein(s) to a preexisting IL-4 DNA-protein complex. Proteins detected in these promoter proximal DNA-protein complexes are likely to be key elements in facilitating stimulation-dependent IL-4 transcription.