The mature T cell receptor (TCR) repertoire is the result of selection events during T cell development. Previous assessment of TCR beta-chain selection with serologic and molecular probes demonstrated both positive and negative selection. Although this work suggested a critical role for the thymus, no direct assessment has been made of the requirement for a thymus in TCR V beta selection. A comparison of TCR V beta expression in four different congenic pairs of normal and nu/nu (athymic) mice indicated that the normal V beta deletions associated with tolerance to self minor lymphocyte stimulating (Mlsc) antigens or to self major histocompatibility complex (MHC)-encoded E alpha E beta products did not occur in most athymic mice. Thus, the thymus has a critical role in mediating self tolerance by negative selection.