The transcription factor Ets-1 critically regulates differentiation and function of T helper (Th) cells. In vitro studies have demonstrated that DNA binding and transcriptional activity of Ets-1 are regulated by phosphorylation. Depending on the site of phosphorylation, Ets-1 function can either be increased or inhibited. In addition, a splice variant lacking several inhibitory phosphorylation sites has been identified, raising the possibility that this splice variant may function differently from the full-length Ets-1. However, it is unclear how the activating and inhibitory phosphorylation events of Ets-1 are coordinated during Th cell activation. Furthermore, the biological consequences of Ets-1 phosphorylation and alternative splicing in regulating the function of Th cells are unknown. We report here that both activating and inhibitory phosphorylation events of Ets-1 occur simultaneously and independently of each other during Th cell activation. We further demonstrate that the effect of Ets-1 phosphorylation is very modest and that full-length Ets-1 and its splice variant are functionally interchangeable in the regulation of cytokine production in Th cells.