The importance of CD28 costimulation to a primary T cell response in vivo was assessed in an adoptive transfer system where a small population of peptide-specific CD4+ TCR transgenic T cells can be physically tracked. Ag-dependent clonal expansion of the transgenic T cells in draining lymph nodes was blocked by cyclosporin A and required a CD28 signal that was completely inhibited by CTLA-4-Ig or a combination of anti-B7-1 and anti-B7-2 mAbs, but not by either Ab alone. In vivo treatment with the combination of anti-B7-1 and anti-B7-2 mAbs also blocked conversion of the Ag-specific T cells to the activated phenotype. In contrast, anti-CTLA-4 Fab greatly enhanced the in vivo clonal expansion of the Ag-specific T cells. These results suggest that Ag-driven proliferation and phenotype conversion of naive CD4+ T cells is dependent on CD28-derived signals and is inhibited by CTLA-4.