Establishment of antigen-specific tolerance among mature T cells has been a long debated, yet poorly understood issue. In this study we have used transgenic mice bearing a class II–restricted TCR specific for the hemmagglutinin of the influenza virus in order to test the behavior of CD4+ T cells upon exposure to antigen in different forms and doses. We first studied the fate of T cells expressing the transgenic TCR (6.5) in double transgenic mice where HA was expressed as a self antigen by hemapoietic cells. In these mice, we found some mature T cells in periphery that had escaped thymic deletion and that showed signs of activation but which were anergic. Mature CD4+6.5+ cells that were transferred into antigen-containing recipients went through an initial phase of expansion after which most cells were deleted and those remaining became unresponsive, as previously described for CD8+ cells. Inducing tolerance in CD4+6.5+ cells in situ in single transgenic mice proved a difficult task: classical protocols using single doses of soluble or deaggregated antigen as well as feeding antigen all failed to induce antigen-specific unresponsiveness. It was only after decreasing cell numbers by CD4 antibody treatment and by repeatedly reintroducing antigen thereafter that unresponsiveness of 6.5+ cells was achieved and maintained. In no case could we observe the appearance of antigen-specific T cells with a Th2 cytokine profile among the remaining cells and therefore conclude that deletion and anergy represent the major mechanisms of tolerance in our studies.