Influenza nucleoprotein (NP) is an important target antigen for influenza A virus cross-reactive cytotoxic T cells (Tc). Here we examine the NP epitope recognized by cloned and polyclonal BALB/c Tc and the genetics of this recognition pattern. We can define NP residues 147-161 as the epitope seen in conjunction with Kd, the only H-2d class I responder allele for NP restriction. H-2d/H-2b F1 mice (C57BL X DBA/2) primed by influenza infection lyse only H-2d target cells treated with peptide 147-161 while H-2b targets are recognized only after treatment with NP residues 365-379 (previously found to be recognized by Db restricted Tc cells). Tc cell recognition of NP peptide 147-161 is entirely dictated by expression of Kd and not by other B10 or C3H background genes of congenic mice. Restriction of a unique NP sequence by each responder class I major histocompatibility complex (MHC) allele suggests that antigen and class I MHC interact for Tc recognition.