A panel of H-2k class II-restricted T helper (Th) clones were established from individual CBA mice intranasally infected with A/X31 influenza virus. In a study of their fine specificity, it was revealed that recognition of hemagglutinin (HA) by certain Th clones was dependent on conformational features of the molecule. Five HA-specific Th clones failed to recognize X31 mutant viruses R19 and R20 each with a His to Arg substitution at position 17 of HA1. This amino acid substitution affects the conformational stability of the molecule. Each of the 5 clones responded to purified HA but failed to recognize tryptic fragments of HA consisting of HA1 residues 28-328 (tops) and the remainder of the virus including residues 1-27 of HA1 (aggregate). A further 20 HA-specific clones responded to R19 and R20 mutants and to HA1 tops. The R19 and R20 negative clones also showed diminished proliferative responses to pH 5-treated X31. Low pH treatment results in an irreversIble conformational change which affects the integrity of the globular head region of the HA trimer. In addition, four of the five clones failed to recognize variant virus Eng-72 which has Arg to Gly substitution at position 208 in the interface antibody-binding region. This region is antigenically and structurally altered in tops and pH 5-treated X31. The results suggest that recognition of HA by certain Th clones is dependent on the three-dimensional structure of the molecule and that Th cells may recognize conformational determinants in addition to primary structure.