Although it is well established that T cells require at least two activation signals, the coordination of primary signaling through the TCR-CD3 complex with costimulatory signals through accessory molecules is incompletely understood. To mimic the signal provided by natural ligand for TCR, we used eight anti-TCR V-region-specific mAbs as well as two anti-TCR-CD3 mAbs, OKT3 and T10B9, to stimulate human peripheral blood T cells in the presence or absence of accessory cells. With accessory cells, only OKT3 in soluble form stimulated T cells, but when mABs were immobilized on plastic, all except the V alpha mAb, F1, induced proliferation. This result suggests that the signaling qualities of TCR V-region-specific mAb may differ from OKT3, which activates through CD3 epsilon. To address this issue, the costimulatory requirements of two V beta-specific mAbs, 1C1 and OT145, were also compared with OKT3 and T10B9 using T cells depleted of accessory cells. The V beta-specific mAb and T10B9 could only be complemented by costimulation through the CD28 molecules, whereas OKT3 was able to synergize with mAb directed not only at CD28, but also CD2 and CD11a. Furthermore, mAb specific for CD80 was able to block activation of T cells in the presence of accessory cells when V beta-specific mAbs were used to activate, whereas anti-CD80 had no effect on activation of T cells by immobilized OKT3. Thus the nature of the signal received through the TCR-CD3 complex, whether TCR alpha beta or CD3 epsilon, may determine the qualitative requirements for costimulation.