Allograft immunity of mice to transplantation antigens was demonstrated by in vitro migration inhibition procedures. Spleen cell suspensions from mice sensitized to histocompatibility antigens by skin graft or i.p. injection of allogeneic spleen cells failed to migrate normally in vitro when incubated with the appropriate spleen cell extracts. Extracts from other lymphoid tissue of the donor mice, including lymph nodes, thymus, and bone marrow, and from nonlymphoid tissue, such as kidney and liver, also resulted in inhibition of migration of spleen cells from sensitized mice. Migration inhibition factor (MIF) was induced in cultures of such cells when incubated with the appropriate spleen cell extracts. The ability of cells derived from other lymphoid organs to respond in the in vitro assay was studied. Lymph node cells from sensitized mice produced MIF when stimulated by alloantigens present in spleen extracts. Thymus and bone marrow cells did not produce MIF under the same conditions. Peritoneal exudate cells from guinea pigs also were effective indicators for the in vitro migration inhibition assay. Either sensitized mouse spleen cells from grafted animals or MIF produced in culture inhibited the migration of the guinea pig macrophages. The degree of inhibition was directly related to the time interval between sensitization of the donor animal and the time of assay. Maximum reactions occurred only during the first 2 weeks after sensitization by skin graft or spleen cell injection. The relationship between the degree of sensitization and in vivo responses was discussed in terms of cellular versus humoral mechanisms.