Several hemagglutinin (HA)-specific T cell hybridomas were generated from five PR8 influenza virus-primed BALB/c mice by fusion with the thymoma BW5147 and were tested for IL 2 (interleukin 2) production in response to a large panel of serologically distinct influenza viruses and HA-protein fragments. The hybridomas (provisionally categorized in the helper T cell lineage) could be placed into three major specificity groups based on their reactivity patterns toward 43 antibody-selected PR8 virus mutants that had been shown previously to differ from parental PR8 virus by defined (usually single) amino acid substitutions in the HA1 polypeptide. Three hybridomas with specificity 1 failed to respond to the PR8 mutant RV6 (Glu115----Lys), two with specificity 2 failed to respond to PR8 mutant DV4 (Ser136----Pro), and two with specificity 3 responded to all antibody-selected PR8 mutants without exception. The three reactivity patterns could be used along with further biochemical evidence to demonstrate the presence of three distinct T cell determinants on the HA1 polypeptide. Priming of mice with one of these T cell determinants in the form of a synthetic peptide induced a T cell subset able to proliferate in vitro in response to HA and intact PR8 virus. Further analysis of the T cell hybridomas for reaction with 17 natural influenza virus isolates of the H1 subtype revealed additional differences in fine nominal antigen specificity among these T cells. However, two pairs of T cells whose members were isolated from different mice exhibited indistinguishable fine nominal antigen specificities. The latter analysis showed that natural antigenic drift (i.e., accumulation of point mutations) in the HA occurring between the years 1934 and 1957 and again between 1977 and 1980 altered two T cell determinants but left one T cell determinant unchanged. The shifts from H1 to H2 or H3 subtypes resulted in the alteration of all three T cell determinants. The present findings suggest that 1) immunization with influenza virus induces HA-specific T cells that recognize determinants different from those seen by anti-HA antibodies, and 2) the HA-specific T cell repertoire of adult virus-primed BALB/c mice is more restricted than the B cell repertoire.