Human T lymphocytes, primed in vitro to influenza virus, were cloned by limiting dilution and expanded using medium containing interleukin 2 and feeder cells. A detailed analysis of the genetic requirements for induction of T-cell proliferation was conducted using a panel of cells from unrelated donors and two families who had previously been extensively phenotyped for HLA region antigens. Clones obtained from a Dw1, Dw3 individual required Dw1,DR1 histocompatibility for successful presentation of viral antigens by antigen-presenting cells. The antigen-presenting ability segregated with HLA-B,D,DR in an informative HLA-A/B recombinant individual. In contrast, some TLCs responded to antigen presented by cells that did not share known HLA antigens, and in one informative family, reactivity did not segregate with HLA. None of the T-cell clones reacted to allogeneic cells in the absence of antigen, suggesting that the TLCs do not bear receptors that recognize both influenza virus and alloantigen. In antibody-blocking studies, Dw1, DR1-restricted clones were blocked by all monoclonal anti-DR framework antibodies. The non-HLA-restricted TLCs were blocked by some, but not all, of the anti-DR framework monoclonal antibodies. These results confirm and extend the role of HLA-D region gene products in antigen presentation and also provide evidence that yet undefined cell interaction products, which may include hybrid structure, are able to participate in antigen-specific proliferative responses by human T cells.