Previous studies have demonstrated that a mAb that recognizes the leukocyte surface Ag V7 inhibits TCR/CD3-dependent T cell activation. In the current study, we demonstrate that in addition to inhibiting T cell proliferation and IL-2 production, anti-V7 blocks tyrosine phosphorylation of TCR/CD3-associated substrates. PMA overcomes this effect, and both PMA and exogenous IL-2 overcome anti-V7-mediated inhibition of T cell proliferation and IL-2 production. T cells stimulated with anti-CD3 in the absence of CD28 or V7 ligation become unresponsive (anergic) to restimulation with anti-CD3; T cells primed in the presence of either anti-V7 or anti-CD28 retain their ability to respond to restimulation with anti-CD3. When T cells are primed in the presence of optimal concentrations of anti-V7 and anti-CD28 Abs, they proliferate normally, indicating that the costimulatory signals generated through CD28 dominate the inhibitory signals generated through V7. However, as the anti-CD28 stimulus is diluted, the V7 effect becomes dominant and proliferation is inhibited. Thus, although both anti-V7 and anti-CD28 Abs prevent anergy, they induce distinct, competing intracellular signals. Wortmannin, which blocks phosphoinositol 3-kinase-dependent signaling, has little effect on V7-mediated inhibition, while herbimycin, an inhibitor of tyrosine kinase, synergizes with anti-V7 to inhibit T cell activation. On the basis of these findings, V7-mediated signals appear to inhibit TCR-dependent tyrosine kinases that are required for IL-2 production and cellular proliferation.