To evaluate the rules of tolerance induction to Mlsa we investigated maturation of T cells in irradiation bone marrow chimeras and thymus transplanted mice expressing the strongly stimulatory Mlsa or the nonstimulatory Mlsb in presence or absence of the permissive MHC class II IE molecule. As shown previously, deletion of V beta 6+, Mlsa reactive T cells required the presence of Mlsa and IE products either on donor cells or on recipient tissue. Additional experiments revealed that tolerance was induced when Mlsa and IE were expressed by distinct cells of the chimera. Similar observations were made in chimeras reconstituted with bone marrow stem cells of two different strains of mice. The presented data confirm earlier evidence that tolerance is induced by cooperation of Mlsa-expressing cells and IE+ Mlsb antigen-presenting cells (APCs). Preliminary results suggest that APCs presenting Mlsa during negative selection of V beta 6+ cells in the thymus are radiosensitive and derived from lymphohemopoietic stem cells.