The cytotoxic T cell response against lymphocytic choriomeningitis virus (LCMV) in BALB/c (H-2d) mice is predominantly directed against a single immunodominant Ld-restricted epitope in the viral nucleoprotein (NP118-126). Here we report that the immunodominance of this peptide can be in part attributed to its very high affinity for Ld class I molecules. By employing motif searches and sensitive MHC class I binding assays, we also identified 5 Kd-binding peptides in the viral nucleoprotein and glycoprotein among 16 Kd motif-fitting peptides. The nucleoprotein and glycoprotein sequences also contained 18 Dd motif-fitting peptides, three of which bound Dd with weak affinity. Two of the Kd-binding peptides, residues 99-108 and residues 283-291 from the viral glycoprotein, are subdominant epitopes. Although these peptides did not sensitize target cells for direct ex vivo killing by primary antiviral CTL, secondary responses against these peptides were readily detected in BALB/c mice after acute LCMV infection. BALB/c mice that had cleared a long-term LCMV infection showed more sustained CTL responses against these subdominant epitopes, suggesting that subdominant responses might play a role in clearance of chronic infections. One of the subdominant epitopes, GP283-291, conferred partial protection against persistent viral infection after peptide vaccination.