While performing a large-scale analysis of mRNA transcripts in the murine thymus, our attention was drawn to the forkhead family transcription factor FKHR. Here we demonstrate that FKHR is expressed in thymocytes, most prominently in those that are undergoing positive selection. Interestingly, FKHR transcripts show a highly regionalized pattern of expression, concentrated in the innermost areas of the medulla. We define the FKHR binding site as (G/C)(A/C)N(G/a)T(A/c)AA(T/c) A(T/g)(T/g)(G/c), a sequence found in the regulatory elements of many genes, including certain that encode molecules crucial for thymocyte differentiation. To study the function of FKHR, we engineered mice expressing a dominant-negative mutant specifically in T cells in a tetracycline-regulatable fashion. In these animals, T cell differentiation appeared quite normal; however, total thymocyte numbers were decreased, owing to reductions in all four of the CD4/CD8 subsets, and incorporation of the thymidine analogue bromo-deoxyuridine was increased, again in all four subsets. These data suggest that, in thymocytes, FKHR may be involved in cell survival and/or cycling.