We studied the impact of the duration of donor cell persistence on CD8⁺ T cell responsiveness after adoptive transfer of antigen‐expressing lymphoid cells. Naive or immunized female mice were treated by adoptive transfer of spleen cells from mice ubiquitously expressing a lymphocytic choriomeningitis virus‐derived cytotoxic T lymphocyte (CTL) epitope (gp33 – 41) either alone or in combination with the male H‐Y antigen providing additional antigenic CTL and T helper cell determinants. Low doses of male spleen cells (or sorted B cells) primed CTL, while high doses of the same cells rendered them unresponsive. CTL unresponsiveness induced by high numbers of male spleen cells was dependent upon prolonged persistence of antigen‐expressing donor cells. Unresponsive CTL reverted to a state of activation when the duration of donor cell chimerism was limited. Memory CTL could be rendered unresponsive if antigen‐expressing donor cells were allowed to persist. These results suggest that, irrespective of the type of antigen‐presenting cell and the functional state of the responding T cell, activation and unresponsiveness can represent two different outcomes critically determined by quantitative and kinetic differences of antigen persistence.