Development of thymocytes can be staged according to the levels of expression of the cell-surface markers CD4, CD8, CD44, CD25 and CD2. Thymocyte development Is regulated by a complex signalling network , one component of which is the GTPase Rho. The bacterial enzyme C3 transferase from Clostridium botulinum selectively ADP-ribosylates Rho in its effector-binding domain and thereby abolishes its biological function [2,3]. To explore the function of Rho in thymocyte development, we previously used the proximal promoter of the gene encoding the Src-family kinase p56lck to make transgenic mice that selectively express C3 transferase in the thymus [4-6]. In these mice, which lack Rho function from the earliest thymocyte stages, thymocyte numbers are reduced by approximately 50- to 100-fold. Here, we describe transgenic mice that express C3 transferase under the control of the locus control region (LCR) of the CD2 gene; this regulatory element drives expression at a later stage of thymocyte development than the lck proximal promoter . In these mice, thymocyte numbers were also reduced by 50- to 100-fold, but unlike the lck-C3 mice, in which the reduction predominantly results from defects In cell survival of CD25+ thymocyte progenitors, the CD2-C3 transgenic mice had a pre-T-cell differentiation block at the CD25+ stage after rearrangement of the T-cell receptor (TCR) chains. Analysis of CD2-C3 mice demonstrated that Rho acts as an intracellular switch for TCRβ selection, the critical thymic-differentiation checkpoint. These results show that Rho-mediated survival signals for CD25+ pre-T cells are generated by the extracellular signals that act on earlier thymocyte precursors and also that temporal cell-type-specific elimination of Rho can reveal different functions of this GTPase in vivo.