CD28 and cytotoxic T-lymphocyte antigen (CTLA)-4 are key coreceptors on the surface of T cells that have opposing effects on T cell activation. Although CD28 enhances proliferation, CTLA-4 markedly inhibits the activation process. These opposing roles are particularly surprising given the structural similarity of the cytoplasmic residues of the two receptors. These include the related CD28(SDYMNM) and CTLA-4(GVYVKM) motifs. In this study, we have directly addressed whether these related motifs may play different roles in the activation process by swapping the CTLA-4(GVYVKM) motif with the CD28(SDYMNM) motif. Remarkably, stable transfectants of the T cell hybridoma DC27.10 showed that substitution of CTLA-4(GVYVKM) was sufficient to convert CTLA-4 from a negative signaling coreceptor to a positive CD28-like coreceptor. CD28(SDYMNM) is therefore sufficient to convey positive signals within CTLA-4. These results demonstrate that CD28(SDYMNM) and CTLA-4(GVYVKM) motifs contain sufficient information to distinguish positive versus negative coreceptor signaling in T cells.