Recent studies of T cell anergy induction have produced conflicting conclusions as to the role of the negative regulatory receptor, CTLA-4. Several in vivo models of tolerance have implicated the interaction of CTLA-4 and its ligands, B7.1 and B7.2, as an essential step in induction of anergy, while results from a number of other systems have indicated that signals from the TCR/CD3 complex alone are sufficient to induce T cell unresponsiveness. One explanation for this disparity is that the requirements for anergy induction depend closely on the details of the system: in vivo vs in vitro, route of stimulus administration, naive vs memory cells, CD4(+) vs CD8(+) cells, etc. To test this possibility, we established an in vivo anergy model using mice transgenic for the 2C TCR on a recombination-activating gene-2-deficient background, that either express or lack the CTLA-4 molecule. This system provides us with a very homogeneous pool of naive Ag-specific CD8(+) T cells, allowing us to control some of the conditions mentioned above. We found that T cells from CTLA-4-deficient mice were anergized by injections of soluble antigenic peptide as efficiently as were CTLA-4-expressing cells. These results indicate that CTLA-4 is not universally required for in vivo T cell anergy induction and may point to distinctions between regulation of peripheral tolerance in CD4(+) and CD8(+) T cells.