Rat thymocytes of the T cell receptorlow (TcRIOW) CD4+8+ subset which is the target of repertoire selection are heterogeneous with respect to expression of the cell interaction (CI) molecules CD2, CD5, CDlldCD18 (LFA-l), CD28 and CD44. We show that this heterogeneity is due to the developmental regulation of these CI molecules during passage through the CD4+8+ compartment, and to up-regulation by TcR engagement. Thus, cohorts of CD4+8+ cells differentiating synchronously in vitro from their direct precursors, the immature CD4-W cells, were homogeneous with regard to CI molecule expression. Upon entry into the CD4+8+ compartment, they expressed relatively high levels of CD2 and CD44, and moderate levels of CD5, CD28 and CDlla. CD2, CD28 and CD44 were slightly down-regulated during the following 2 days, whereas CD5 slightly increased and CDlla remained constant. TcR stimulation using immobilized monoclonal antibodies resulted in rapid and dramatic up-regulation of CD2, CD5 and CD28 and, to a lesser extent, of CDlla and CD44. Finally CD53, a triggering structure absent from unstimulated CD4+8+ thymocytes was also rapidly induced by TcR stimulation. Inclusion of interleukin (1L)-2, IL-4, or IL-7 in this in vitro differentiation system did not affect the levels of CI molecules studied. Since the high levels of CI molecules induced by TcR-stimulation correspond to those found in vivo onTcRintermediate thymocytes known to be undergoing repertoire selection, these results suggest that upregulation of CI molecules by TcR engagement provides a mechanism by which thymocytes that have entered the selection process gain preferential access to further interactions with stromal and lymphoid cells in the thymus.