Abstract BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.