The progression of T cells from a quiescent or resting state to fully activated, proliferating cells is a crucial step in the initiation of an immune response. We have developed an in vitro system to study the requirements for triggering or hindering this pathway by using naive T cells derived from T-cell antigen receptor alpha beta transgenic animals and peptide-major histocompatibility (MHC) complexes coated on plates. Whereas previously stimulated T cells require only peptide-MHC complexes to produce interleukin 2 (IL-2), naive cells require at least one additional signal, which can be provided by either an anti-CD28 antibody or the protein kinase C stimulant phorbol 12-myristate 13-acetate. In contrast, the anti-CD28 antibody augments IL-2 production by primed T cells but is not required, and phorbol 12-myristate 13-acetate has no discernable effect. Thus we find that native T cells have significantly more stringent requirements for IL-2 production than primed cells and that this fits well with previous observations in other in vitro systems as well as in vivo models of autoimmunity. We also find that peptide-MHC complex stimulation of naive T cells, together with exogenous IL-2, is sufficient to convert these cells to primed T cells in vitro in 2 days, as assayed both by surface marker analysis and stimulation requirements. Taken together with the above results, this suggests that the activation of primary T cells requires at least two signals and that IL-2 produced by naive T cells in vivo may act in an autocrine fashion to allow them to proliferate and differentiate.