CD27 is a lymphocyte-specific member of the TNF receptor family and has a TNF-related transmembrane ligand, CD70. The CD27/CD70 receptor-ligand pair cooperates with the TCR in the regulation of the peripheral T cell response. The study presented here reveals that CD27 may play a similar role in thymic pre-T cell development. We have previously cloned the cDNA encoding murine CD27, prepared specific mAbs and observed that murine CD27 is expressed on virtually all thymocytes, with the exception of a subpopulation of CD4-8- precursor T cells. It is shown here that induction of murine CD27 expression occurs at the transition from the CD4-8-25+ to the CD4-8-25- precursor T cell stage and is regulated by the pre-TCR. Therefore, we investigated whether CD27 contributes to pre-TCR-mediated thymocyte development. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombination activating gene (RAG)-deficient mice. This in vivo anti-CD3 epsilon mAb treatment induces an about fifty fold numerical expansion of CD4-8-25+ thymocytes and their differentiation to the CD4+8+25- stage. Co-injection of anti-CD27 mAb inhibited the CD3-mediated expansion and differentiation of the CD4-8-25+ precursor population. Also, injection of anti-CD27 mAb in TCR alpha-/- mutant mice led to a reduction in the absolute number of CD4+8+25- thymocytes. We present evidence that in these in vivo systems, anti-CD27 mAb inhibits CD27-ligand interaction. Therefore, we conclude that CD27 may contribute to normal murine T cell development by synergizing with the pre-TCR-mediated signal.