c Diacylglycerol kinase ␣ (DGK␣) regulates diacylglycerol levels, catalyzing its conversion into phosphatidic acid. The ␣ isoform is central to immune response regulation; it downmodulates Ras-dependent pathways and is necessary for establishment of the unresponsive state termed anergy. DGK␣ functions are regulated in part at the transcriptional level although the mechanisms involved remain poorly understood. Here, we analyzed the 5= end structure of the mouse DGK␣ gene and detected three binding sites for forkhead box O (FoxO) transcription factors, whose function was confirmed using luciferase reporter constructs. FoxO1 and FoxO3 bound to the 5= regulatory region of DGK␣ in quiescent T cells, as well as after interleukin-2 (IL-2) withdrawal in activated T cells. FoxO binding to this region was lost after complete T cell activation or IL-2 addition, events that correlated with FoxO phosphorylation and a sustained decrease in DGK␣ gene expression. These data strongly support a role for FoxO proteins in promoting high DGK␣ levels and indicate a mechanism by which DGK␣ function is downregulated during productive T cell responses. Our study establishes a basis for a causal relationship between DGK␣ downregulation, IL-2, and anergy avoidance.T he diacylglycerol kinases (DGK) phosphorylate diacylglycerol (DAG) into phosphatidic acid (PA), modulating the levels of these two lipid second messengers, which have several key functions in cells. DAG propagates signals by membrane recruitment of cytosolic proteins containing C1 domains, such as protein kinase C and D (PKC and PKD, respectively), the Ras-guanine nucleotide exchange factor (GEF) RasGRP1, and the Rac-GTPaseactivating protein (GAP) chimaerins (3). DAG deregulation is linked to tumorigenesis, metastasis, diabetes, heart disease, and altered immune responses (9,13,45,53). PA binds and activates proteins involved in cell growth, survival, vesicular trafficking, and cytoskeletal remodeling, and its altered metabolism is also linked to disease onset (7,14,40). Interest in the DGK as key modulators of DAG and PA function has increased in recent years as better understanding of DGK regulatory mechanisms offers opportunities for the development of novel strategies to modulate lipid metabolism for therapeutic purposes (for reviews, see references 32 and 44).DGK function attracted special attention following the characterization of its role in T lymphocyte activation. Productive activation of T lymphocytes requires the integration of the pathways regulated by Ras/mitogen-activated protein kinase (MAPK)/AP1 and Ca 2ϩ /nuclear factor of activated T cells (NFAT). Failure to trigger an adequate balance of these signals, due, for example, to lack of costimulation, drives T cells into a nonresponsive state termed anergy, in which cells survive for long periods in the absence of proliferation (1). DGK␣ is a type I DGK particularly abundant in thymus and mature T lymphocytes (55), and early studies showed its function as a negative modulator of the Ras/ MAPK pathway. DGK␣ limits...