We and others previously reported that cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates the severity of peptide-induced experimental autoimmune encephalomyelitis (EAE) in mouse strains that are inherently susceptible to the disease. In this report, we show that CTLA-4 engagement also controls disease susceptibility in BALB/c mice, a strain considered to be resistant to EAE induction. Although immunization of BALB/c mice with syngeneic spinal cord homogenate or an I-A(d)-binding myelin peptide antigen failed to result in EAE, immunization with either antigen preparation in conjunction with anti-CTLA-4 resulted in both clinical and histological EAE. CTLA-4 blockade also resulted in a preferential increase in the frequency of antigen-specific T cells secreting IFN-gamma. We conclude that CTLA-4 controls susceptibility in BALB/c mice by limiting the expansion of autoreactive T cells present in the periphery, suggesting a mechanism whereby CTLA-4 contributes to the maintenance of peripheral T cell tolerance to self antigens.