Co-stimulation is a fundamental principle of T-cell activation. In addition to TCR engagement, the interaction between CD80 and/or CD86 with CD28 and/or CTLA-4 receptors is important for T-cell activation and tolerance. While the requirement of co- stimulation is clearly established, the exact molecular mechanism is unknown. We show that T-cell proliferation and killing capacity of CD8+ T-effector cells were dramatically enhanced by CD86 but only slightly by CD80 co-stimulation. This enhancement was paralleled by a large increase of Ca2+ influx through CD86 but only a slight one through CD80 co-stimulation. The differences in Ca2+ influx were eliminated by the application of 10 mM 2-APB, which has been shown to enhance STIM2- dependent Ca2+ entry while reducing STIM1-dependent one. Down-regulation of STIM1 by siRNA reduced co-stimulation dependent Ca2+ entry. We conclude that CD86 and CD80 regulate the immune response by modulating Ca2+ influx, perhaps by differential activation of STIM1 and STIM2.