We examined how cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates heterogeneous CD4⁺ T cell responses by using experimental autoimmune encephalomyelitis (EAE), a CD4⁺ T cell-mediated disease that is subject to regulation by CTLA-4. Disease incidence and severity were used as measures of in vivo CD4⁺ T cell responses. The frequency, cytokine production, and reactivity of primed T cells were determined from animals immunized with proteolipid protein (PLP)-139–151 (disease agonist), PLP-Q (disease antagonist), or both peptides, and treated with control or anti-CTLA-4 antibody to analyze the responding population. CTLA-4 blockade exacerbated disease in PLP-139–151-primed animals and overcame disease antagonism in coimmunized animals, but did not permit disease induction in PLP-Q-primed animals. Experimental autoimmune encephalomyelitis enhancement was associated with increased frequencies of cytokine-producing cells and increased ratios of IFN-γ to IL-4 secretors responsive to PLP-139–151. Priming with PLP-Q elicited IL-4 and IL-2, but not IFN-γ secretors cross-reactive with PLP-139–151. Strikingly, CTLA-4 blockade was found to decrease rather than increase the frequencies of cross-reactive IL-4 and IL-2 secretors. Thus, CTLA-4 engagement limits the size, but increases the breadth, of reactivity of a primed pool of CD4⁺ T cells, consequently regulating its function.