The lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II ligand evolutionarily related to CD4, is expressed exclusively in activated T and NK lymphocytes and seems to play a role in regulating the evolving immune response. We first determined that surface LAG-3 expression on activated human T cells is upregulated by certain cytokines (IL-2, IL-7, IL-12) and not by others (IL-4, IL-6, IL-10, TNF-alpha, TNF-beta, IFN-gamma). Surface LAG-3 expression correlated with intracellular IFN-gamma production in both CD4+ and CD8+ T-cell subsets. We then analyzed the 5' transcription control sequences of LAG-3. A DNase I hypersensitive site induced in T cells following cellular activation was found in the region including the transcriptional start site, showing that DNA accessibility is a mechanism which restricts LAG-3 expression to activated T cells. Transcription is initiated at three sites. A GC box, 80 base pairs (bp) upstream of the major transcription start site, forms a minimal promoter which is regulated by two upstream regions containing positive and negative regulatory elements with multiple protein binding sites as shown by footprinting analysis. In particular, a GATA/c-Ets motive was identified in a short segment homologous to the mouse CD4 distal enhancer, suggesting that LAG-3, which is embedded in the CD4 locus, may be controlled by some CD4 regulatory elements. Finally, a 100 bp region downstream of the transcription start site was shown to be involved in the cell-specific control of LAG-3 expression. Understanding this highly regulated expression may help to determine the intriguing role of this activation-induced MHC class II ligand.