The strain-specific unresponsiveness to H-2 incompatible skin allografts induced by treatment of adult mice with single inoculations of donor strain liver extract and Bordetella pertussis vaccine, as well as three doses of antilymphocyte serum, has been investigated by several in vivo and in vitro methods, with a view to elucidating it mechanism. Lymphoid cells from mice with long surviving skin grafts were found to be reactive in graft-versus-host assays (as measured by splenomegaly or popliteal lymph node enlargement), and mixed lymphocyte culture tests gave positive results. Attempts to cause lethal runting of F1 hybrid mice injected at birth with spleen cells from unresponsive mice gave variable results. However, the injection of F1 hybrid cells into the footpads of unresponsive animals failed to elicit a significant host-versus-graft response. Although lymphoid cells from unresponsive animals did not include detectable numbers of cytotoxic cells, such cells could be generated by previous in vitro mixed lymphocyte culture stimulation or, to some degree, by the injection of the animals with F1 hybrid cells. Attempts to prevent mixed lymphocyte culture stimulation or cytotoxicity with serum from unresponsive mice failed at the serum concentrations used. The data indicate that long-term unresponsiveness in this system is maintained by the production in the hosts of factors that interfere with the cell-mediated response.