The Ets and AP-1 families of transcription factors bind distinct DNA elements and subserve diverse functions in multiple lymphoid and nonlymphoid cell types. Functionally important Ets and AP-1 binding sites have been identified in a large number of enhancer elements, suggesting important cooperative interactions between these two families of transcription factors. In this report, we have demonstrated a direct physical interaction between Ets and AP-1 proteins both in vitro and in activated human T cells. This interaction is mediated by the binding of the basic domain of Jun to the Ets domain of Ets proteins. Jun, in association with Ets, is capable of interacting with Fos family members to form a trimolecular protein complex. The physical association between Ets-1 and AP-1 proteins is required for the transcriptional activity of enhancer elements containing adjacent Ets and AP-1 binding sites. We conclude that direct physical interactions between Ets and AP-1 transcription factors play an important role in regulating mammalian gene expression.