Ly-6A.2 is a surface protein on T cells that may play a role in lymphocyte activation. The regulation of Ly-6A.2 expression during T cell lymphopoiesis has been intriguing. It is one of the earliest markers expressed on pluripotent hemopoietic stem cells and is present on both primitive and mature T cells, but its expression is extinguished in the thymus during key developmental stages. To determine whether Ly-6A.2 is active on developing T cells, as well as the significance of its developmental regulation, Ly-6A.2 was expressed throughout T cell development under control of the T cell-specific human CD2 enhancer in transgenic mice. The constitutive overexpression of Ly-6A.2 in vivo led to a marked impairment in the generation of thymocytes. Development was arrested at the time in thymic development when Ly-6A.2 expression is normally turned off. These results indicate that the regulated expression of Ly-6A.2 in thymocytes may be important for normal development. Moreover, these findings demonstrate that Ly-6A.2 is active in the thymic microenvironment.