Lymphocytic choriomeningitis virus (LCMV) is known to induce strong, polyclonal cytotoxic T-lymphocyte (CTL) responses. Using a set of variant peptides derived from the major CTL epitope of LCMV, we analyzed the functional fine specificity of the LCMV-specific CTL response. During the primary response, almost all the tested peptides were recognized. In contrast, the secondary response was purged of all minor cross-reactivities and very few peptides were significantly recognized. This study is the first demonstration of the functional maturation of a T-cell response and has important clinical and biological implications.