The cytotoxic T-lymphocyte (CTL) response to lymphocytic choriomeningitis virus infection determines the outcome of infection. Here we show that this response in BALB/c mice (H-2d), when analyzed both at the primary CTL level and using CTL clones, is predominantly monospecific. The vast majority of CTL have a common specificity for a single epitope in the virus nucleoprotein, which can be minimally identified by amino acids GVYMG. This epitope is presented by the Ld class I glycoprotein. We used these data to design a subunit CTL vaccine, whose effectiveness is demonstrated in the accompanying report (L. S. Klavinskis, J. L. Whitton, and M. B. A. Oldstone, J. Virol. 63:4311-4316, 1989). Further analysis indicates that, while CTL clones share a common minimal epitope, they differ in their ability to recognize cells infected with a related but distinct strain of lymphocytic choriomeningitis virus. Studies on the molecular nature of CTL cross-reactivity indicate that CTL induced by similar sequences may cross-react in a unidirectional manner. These novel observations suggest that CTL vaccines, to achieve optimal effectiveness, should not simply include virus sequences which will yield a CTL response; the immunizing sequences should also be selected to ensure that the fine specificities of the induced CTL are such that they maximize the chance of recognizing serotypically diverse strains.