Inhibitory killer Ig-like receptors (KIR), expressed by human natural killer cells and effector memory CD8(+) T-cell subsets, bind HLA-C molecules and suppress cell activation through recruitment of the Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1). To further analyze the still largely unclear role of inhibitory KIR receptors on CD4(+) T cells, KIR2DL1 transfectants were obtained from a CD4(+) T-cell line and primary cells. Transfection of CD4(+) T cells with KIR2DL1 dramatically increased the T-cell receptor (TCR)-induced production of interleukin-2 independently of ligand binding but inhibited TCR-induced activation after ligation. KIR-mediated costimulation of TCR activation involves intact KIR2DL1-ITIM phosphorylation, SHP-2 recruitment, and PKC- phosphorylation. Synapses leading to activation were characterized by an increase in the recruitment of p-Tyr, SHP-2, and p-PKC-, but not of SHP-1. Interaction of KIR2DL1 with its ligand led to a strong synaptic accumulation of KIR2DL1 and the recruitment of SHP-1/2, inhibiting TCR-induced interleukin-2 production. KIR2DL1 may induce 2 opposite signaling outputs in CD4(+) T cells, depending on whether the KIR receptor is bound to its ligand. These data highlight unexpected aspects of the regulation of T cells by KIR2DL1 receptors, the therapeutic manipulation of which is currently being evaluated.