APC-associated B7 and ETC-1/B7-2 are two major costimulatory molecules for full activation of T lymphocytes during auto- and allogeneic immune responses. In this report, we further examine the role of the two molecules in murine CD4+ T cell activation and anergy development. As suggested in antibody blocking studies, optimal activation of CD4+ T cells in response to anti-CD3 stimulation requires collaborative signaling through the two molecules. Simultaneous blockade of B7 and ETC-1/B7-2 renders CD4+ T cells unresponsive to anti-CD3 restimulation. PCR analysis and cytokine reconstitution studies show that the observed unresponsiveness is correlated to a significant reduction of Th1-type cytokine production, suggesting B7 and ETC-1/B7-2-mediated costimulatory signaling may be specifically active in regulation of the function of the Th1 subset.