The fine specificity of virus recognition by influenza A/PR8/34(H1N1)-specific cytolytic T lymphocyte (CTL) clones was analyzed with the use of a panel of syngeneic target cells infected with five heterologous influenza A strain viruses. Forty-five H-2 D-end-restricted CTL clones from B10.A(5R) responders (Dd,Ld) demonstrated 14 different patterns of recognition. Many of these clonotypes were able to distinguish between closely related viruses of the same subtype. Such discriminatory capacity, however, was often accompanied by cross-reactivity against a distantly related viral subtype. This supports the contention that virus-specific CTL see different structures than do virus-specific antibodies. A similar analysis of the fine specificity of 60 Db-restricted clones from C57BL/6 responders was performed. The vast majority of this response was composed of clonotypes not observed in the B10.A(5R) response. In addition, the hierarchy of relatedness between the virus strain used for immunization and the various heterologous viruses was different in C57BL/6 and B10.A(5R). In contrast, the D-end-restricted response of Balb/c (Dd,Ld) demonstrated clonotypes similar to those found in B10.A(5R). These data suggest that determinant recognition in an anti-viral CTL response is a function of the H-2 restricting elements, and this is discussed in the context of determinant selection by class I molecules.